Reagents and methods for analysis of proteins and metabolites targeted by covalent probes

ABSTRACT

The present application relates to mass spectrometry methods for use in identifying proteins or other biomolecules which are bound irreversibly by test compounds.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.16/461,486, filed on May 16, 2019, which is a national phase filingunder 35 U.S.C. § 371 of International application numberPCT/US2017/063443, filed Nov. 28, 2017, which claims the benefit of U.S.Provisional Application Ser. No. 62/427,042, filed Nov. 28, 2016. Thedisclosures of the prior applications are incorporated herein byreference in their entirety.

SEQUENCE LISTING

This application contains an Amended Sequence Listing that has beensubmitted electronically in computer readable form named2211C01US_Amended_Seq_Listing_28APR2023. The ASCII file, created on Apr.24, 2023, is 3750 bytes in size. The material in the ASCII file ishereby incorporated by reference in its entirety.

TECHNICAL FIELD

This disclosure relates to mass spectrometry-based identification ofproteins or other biomolecules which are bound irreversibly by testcompounds.

BACKGROUND

The thiol side-chain of cysteine is subject to numerous endogenous(e.g., enzymatic or metabolic) and environmentally-mediated chemicalmodifications. In addition, there is renewed interest in developingsmall molecules which exert therapeutic effect via covalent modificationof cysteine residues in target proteins. In cases involving endogenous(e.g., cellular) target discovery, the specific cysteine residue, oroften the protein itself, which is modified by the covalent probe is notknown. Even when the target is known, the landscape of off-targetmolecules remains difficult to identify and may represent a confoundingvariable in determining (poly)pharmacology, therapeutic window,potential side effects, etc. Similarly, for any established covalentprobe-target combination, it is difficult to quantify the fraction ofendogenous target molecule that is bound by the covalent probe. Giventhe biochemical complexity and dynamic range of mammalian proteomes,high-throughput characterization of small molecule covalent probesremains enormously challenging.

Several approaches have been developed as surrogates for theidentification of proteins or other biomolecules covalently modified bysmall molecule probes. In one method, a small molecule of interest issynthesized with an affinity tag (e.g., biotin) or a biorthogonalreactive group (e.g., alkyne) which is subsequently used as a “handle”to enrich covalently-bound protein targets from complex biologicalmixtures. The addition of these moieties can disrupt the bindingkinetics and/or activity of the native probe and may also negate cellplasma membrane permeability, requiring incubation of the probe inprotein lysate instead of directly in live cells. A second approach usesbroad-activity probes, built around iodoacetamide, for example, whichare used in a ‘competition format’ with the small molecule inhibitor ofinterest. In this case, the readout is “indirect,” meaning that proteinsnot detected, or detected with significantly reduced abundance, areassumed to be modified by the experimental inhibitor, and henceless-available for labeling by the broad-activity probes. The indirectassay may not include an enrichment step, and therefore may be furtherlimited by the stochastic and abundance-biased sequencing inherent tomass spectrometry-based identification. A more recent method relies onthe ability of small molecule probes to impart increased thermalstability to their targets. This paradigm utilizes native probes in livecells with subsequent thermal cycling of lysates. The assumption is thatnon-bound protein targets will precipitate from solution, while modifiedproteins will remain solubilized and available for processing andidentification by mass spectrometry-based techniques. The variables forprobe-mediated thermal stability are not completely understood. As aresult, this assay format is subject to high or indeterminatefalse-positive/-negative rates.

SUMMARY

The present application provides, inter alia, an analytical method,comprising:

-   -   i) contacting a test compound with a polypeptide to form a test        compound-polypeptide conjugate;    -   ii) analyzing the test compound-polypeptide conjugate using a        mass spectrometry assay;    -   iii) detecting one or more thiolated ions, or derivative ions        thereof, produced in the mass spectrometry assay; and    -   iv) identifying that the test compound irreversibly bonds to the        polypeptide based on the detection of the one or more thiolated        ions, or derivative ions thereof, in the mass spectrometry        assay.

In some embodiments, the method comprises:

-   -   i) contacting a test compound with a polypeptide to form a test        compound-polypeptide conjugate;    -   ii) analyzing the test compound-polypeptide conjugate using a        mass spectrometry assay;    -   iii) detecting one or more thiolated ions, or derivative ions        thereof, produced in the mass spectrometry assay; and    -   iv) identifying that the test compound irreversibly bonds to the        polypeptide based on the detection of the one or more thiolated        ions, or derivative ions thereof, in the mass spectrometry        assay.

In some embodiments, the compound-polypeptide conjugate comprises one ormore thioether bonds between the test compound and the polypeptide. Insome embodiments, the irreversible bond is an irreversible covalentbond.

In some embodiments, step i) comprises contacting the test compound andthe polypeptide in the presence of a first solvent component. In someembodiments, the first solvent component is DMSO. In some embodiments,step i) further comprises contacting the compound and the polypeptide inthe presence of a buffer agent. In some embodiments, the buffer agent istriethylammonium bicarbonate. In some embodiments, step i) is performedat a temperature of about 4° C. to about 65° C. In some embodiments,step i) is performed for about 1 second to about 16 hours. In someembodiments, step i) is performed using a molar excess of the testcompound compared to the polypeptide. In some embodiments, the molarratio of the test compound to the polypeptide is from about 1:1 to about100:1.

In some embodiments, the method further comprises contacting the testcompound-polypeptide conjugate with an acid in the presence of a secondsolvent component prior to performing the mass spectrometry assay ofstep ii). In some embodiments, the acid is an organic acid. In someembodiments, the acid is acetic acid. In some embodiments, the secondsolvent component comprises acetonitrile. In some embodiments, thesecond solvent component further comprises water.

In some embodiments, the method further comprises digesting the testcompound-polypeptide conjugate prior to the performing the massspectrometry assay of step ii). In some embodiments, the digestingcomprises reacting the test compound-polypeptide conjugate with trypsinin the presence of a third solvent component. In some embodiments, thethird solvent component comprises aqueous ammonium bicarbonate.

In some embodiments, the polypeptide comprises one or more amino acidsresidues comprising at least one sulfur atom. In some embodiments, thepolypeptide comprises one or more cysteine residues. In someembodiments, the test compound is identified as irreversibly bonding toone or more cysteine residues of the polypeptide. In some embodiments,the test compound comprises one or more acrylamide groups. In someembodiments, the test compound comprises one or more acrylamide groups,dimethylamino acrylamide groups, iodoacetamide groups, chloroacetamidegroups, maleimide groups, or reactive C—X bonds, wherein X is a halogen.In some embodiments, the test compound is isotopically labeled withheavy isotopes of carbon, oxygen, nitrogen, sulfur, phosphorous,chlorine, bromine, or hydrogen.

In some embodiments, the test compound is identified as a kinaseinhibitor or a deubiquitinase inhibitor. In some embodiments, the testcompound is identified as a kinase inhibitor. In some embodiments, thetest compound is selected from the group consisting of JNK-IN-7,HBX-19818, MI-2, TL10-201, THZ531, THZ1, QL-47, ibrutinib, andneratinib. In some embodiments, the test compound is selected from thegroup consisting of JNK-IN-7, HBX-19818, MI-2, TL10-201, THZ531, THZ1,QL-47, TL11-113, ibrutinib, and neratinib.

In some embodiments, the test compound is chemically modified tofacilitate affinity-based enrichment of test compound polypeptideconjugates. In some embodiments, chemical-modification of the testcompound comprises addition of an affinity tag such as apeptide-epitope, biotin, or desthiobiotin. In some embodiments, thechemical-modification of the test compound comprises addition of abio-orthogonal moiety such as an alkyne or azide.

In some embodiments, a reagent having broad thiol reactivity and highyield of thiolated ions, or derivative ions thereof, is used to measurethe binding stoichiometry of the test compound to its target. In someembodiments, the reagent with broad thiol reactivity and high yield ofthiolated ions, or derivative ions thereof, is selected from the groupconsisting of:

-   2-iodo-1-morpholinoethan-1-one;-   1-((2R,6R)-2,6-dimethylmorpholino)-2-iodoethan-1-one;-   1-((2R,6S)-2,6-dimethylmorpholino)-2-iodoethan-1-one;-   1-(2,2-dimethylmorpholino)-2-iodoethan-1-one;-   1-(3,5-dimethylmorpholino)-2-iodoethan-1-one;-   1-(hexahydrocyclopenta[b][1,4]oxazin-4(4aH)-yl)-2-iodoethan-1-one;-   1-(2,3-dihydro-4H-benzo[b][1,4]oxazin-4-yl)-2-iodoethan-1-one;-   N-(4-cyanophenyl)-2-iodoacetamide;-   N-(2,5-dimethylphenyl)-2-iodoacetamide;-   N-(2,5-dimethoxyphenyl)-2-iodoacetamide;-   2-iodo-N-phenylacetamide;-   2-iodo-N-(p-tolyl)acetamide;-   2-iodo-1-(4-methylpiperazin-1-yl)ethan-1-one 2,2,2-trifluoroacetate;-   N-(furan-2-ylmethyl)-2-iodoacetamide;-   2-iodo-N-(1-methyl-1H-imidazol-4-yl)acetamide; and-   N-ethylmaleimide.

In some embodiments, the reagent having broad thiol reactivity isisotopically labeled with heavy isotopes of carbon, oxygen, nitrogen,sulfur, phosphorous, chlorine, bromine, or hydrogen.

In some embodiments, the polypeptide is a protein or a protein fragment.In some embodiments, the polypeptide is a protein fragment comprisingfrom about 10 to about 30 amino acid residues.

In some embodiments, the polypeptide is a kinase, a kinase fragment, adeubiquitinase, or a deubiquitinase fragment. In some embodiments, thepolypeptide is a kinase or deubiquitinase selected from the groupconsisting of JNK2, JAK3, CDK7, CDK12, TAK1, ITK, USP-7, and EGFR, or afragment thereof.

In some embodiments, the polypeptide is a kinase or a kinase fragment.In some embodiments, the polypeptide is a kinase selected from the groupconsisting of JNK2, JAK3, CDK7, CDK12, ITK, USP-7, and EGFR, or afragment thereof. In some embodiments, the polypeptide is a kinasefragment comprising from about 10 to about 30 amino acid residues.

In some embodiments, the polypeptide comprises an amino acid sequencehaving at least 90% sequence identity to a sequence selected from thegroup consisting of:

(SEQ ID NO: 1) L-M-D-A-N-L-C-Q-V-I-Q-M-E; (SEQ ID NO: 2)L-V-M-E-Y-L-P-S-G-C-L-R; (SEQ ID NO: 3)M-A-P-P-D-L-P-H-W-Q-D-C-H-E-L-W-S-K; (SEQ ID NO: 4)H-G-C-L-S-D-Y-L-R-S-Q-R-G-L-F-A-A-E; (SEQ ID NO: 5)Y-F-S-N-R-P-G-P-T-P-G-C-Q-L-P-R-P-N-C-P-V-E-T-L-K; (SEQ ID NO: 6)G-C-L-L-D-Y-V-R; (SEQ ID NO: 7) F-G-L-C-S-G-P-A-D-T-G-R;(SEQ ID NO: 8; sL = ¹⁵N-1, ¹³C-6 leucine) Y-M-A-N-G-C-L-sL-N-Y-L-R;(SEQ ID NO: 9) I-C-D-F-G-T-A-C-D-I-Q-T-H-M-T-N-N-K; and (SEQ ID NO: 10)Y-F-S-N-R-P-G-P-T-P-G-C-Q-L-P-(13C6-15N4)R-P-N-C- P-V-E-T-L-K.

In some embodiments, the polypeptide comprises an amino acid sequencehaving at least 90% sequence identity to a sequence selected from thegroup consisting of:

(SEQ ID NO: 1) L-M-D-A-N-L-C-Q-V-I-Q-M-E; (SEQ ID NO: 2)L-V-M-E-Y-L-P-S-G-C-L-R; (SEQ ID NO: 3)M-A-P-P-D-L-P-H-W-Q-D-C-H-E-L-W-S-K; (SEQ ID NO: 4)H-G-C-L-S-D-Y-L-R-S-Q-R-G-L-F-A-A-E; (SEQ ID NO: 5)Y-F-S-N-R-P-G-P-T-P-G-C-Q-L-P-R-P-N-C-P-V-E-T-L-K; (SEQ ID NO: 6)G-C-L-L-D-Y-V-R; (SEQ ID NO: 7) F-G-L-C-S-G-P-A-D-T-G-R; and(SEQ ID NO: 8) Y-M-A-N-G-C-L-sL-N-Y-L-R.

The present application further provides an analytical method,comprising:

-   -   i) contacting a first mixture comprising one or more test        compounds with a second mixture comprising one or more        polypeptides to form a third mixture comprising one or more        compound-polypeptide conjugates, wherein each of the        compound-polypeptide conjugates comprise one or more thioether        bonds;    -   ii) analyzing the third mixture using a mass spectrometry assay;    -   iii) detecting one or more thiolated ions, or derivative ions        thereof, produced in the mass spectrometry assay; and    -   iv) identifying that one or more of the test compounds binds        irreversibly to one or more of the polypeptides based on the        detection of the one or more thiolated ions, or derivative ions        thereof, in the mass spectrometry assay.

The present application further provides an analytical method,comprising:

-   -   i) contacting a first mixture comprising one or more test        compounds with a second mixture comprising one or more        polypeptides to form a third mixture comprising one or more        compound-polypeptide conjugates, wherein each of the        compound-polypeptide conjugates comprise one or more thioether        bonds;    -   ii) analyzing the third mixture using a mass spectrometry assay;    -   iii) detecting one or more thiolated ions produced in the mass        spectrometry assay; and    -   iv) identifying that one or more of the test compounds binds        irreversibly to one or more of the polypeptides based on the        detection of the one or more thiolated ions in the mass        spectrometry assay.

In some embodiments, the method further comprises digesting the testcompound-polypeptide conjugate prior to performing the mass spectrometryassay of step ii). In some embodiments, the digesting comprises reactingthe test compound-polypeptide conjugate with trypsin in the presence ofa third solvent component. In some embodiments, the third solventcomponent comprises aqueous ammonium bicarbonate.

In some embodiments, each of the one or more test compound-polypeptideconjugates comprises one or more thioether bonds between the testcompound and the polypeptide. In some embodiments, each of thepolypeptides comprises one or more cysteine residues. In someembodiments, each of the test compounds is identified as irreversiblybonding to one or more cysteine residues of at least one of the one ormore polypeptides. In some embodiments, each of the test compoundscomprises one or more thiol-reactive groups including, but not limitedto, acrylamide groups, dimethylamino acrylamide groups, iodoacetamidegroups, chloroacetamide groups, maleimide groups, or reactive C—Xgroups, wherein X is a halogen. In some embodiments, each of the testcompounds are isotopically labeled with heavy isotopes of carbon,oxygen, nitrogen, sulfur, phosphorous, chlorine, bromine, or hydrogen.In some embodiments, each of the test compounds comprises one or moreindependently selected acrylamide groups. In some embodiments, the testcompound is chemically modified to facilitate affinity-based enrichmentof test compound polypeptide conjugates. In some embodimentschemical-modification of the test compound comprises addition of anaffinity tag such as a peptide-epitope, biotin, or desthiobiotin. Insome embodiments the chemical-modification of the test compoundcomprises addition of a bio-orthogonal moiety such as an alkyne orazide.

In some embodiments, one or more of the test compounds is identified asa kinase inhibitor or a deubiquitinase inhibitor. In some embodiments,one or more of the test compounds is identified as a kinase inhibitor.In some embodiments, at least one of the test compounds is selected fromthe group consisting of JNK-IN-7, TL10-201, THZ531, THZ1, QL-47,ibrutinib, neratinib and TL11-113. In some embodiments, at least one ofthe test compounds is selected from the group consisting of JNK-IN-7,TL10-201, THZ531, THZ1, QL-47, ibrutinib, and neratinib. In someembodiments, each of the one or more polypeptides is a protein or aprotein fragment. In some embodiments, the polypeptide is a proteinfragment comprising from about 10 to about 30 amino acid residues. Insome embodiments, each of the one or more polypeptides is a kinase, akinase fragment, a deubiquitinase or a deubiquitinase fragment. In someembodiments, each of the one or more polypeptides is a kinase or akinase fragment.

In some embodiments, at least one of the polypeptides is a kinase ordeubiquitinase selected from the group consisting of JNK2, JAK3, CDK7,CDK12, ITK, USP-7, TAK1, and EGFR, or a fragment thereof.

In some embodiments, at least one of the polypeptides is a kinaseselected from the group consisting of JNK2, JAK3, CDK7, CDK12, ITK,USP-7, and EGFR, or a fragment thereof.

In some embodiments, the polypeptide is a kinase fragment comprisingfrom about 10 to about 30 amino acid residues. In some embodiments, atleast one of the polypeptides comprises an amino acid sequence having atleast 90% sequence identity to a sequence selected from the groupconsisting of:

(SEQ ID NO: 1) L-M-D-A-N-L-C-Q-V-I-Q-M-E; (SEQ ID NO: 2)L-V-M-E-Y-L-P-S-G-C-L-R; (SEQ ID NO: 3)M-A-P-P-D-L-P-H-W-Q-D-C-H-E-L-W-S-K; (SEQ ID NO: 4)H-G-C-L-S-D-Y-L-R-S-Q-R-G-L-F-A-A-E; (SEQ ID NO: 5)Y-F-S-N-R-P-G-P-T-P-G-C-Q-L-P-R-P-N-C-P-V-E-T-L-K; (SEQ ID NO: 6)G-C-L-L-D-Y-V-R; (SEQ ID NO: 7) F-G-L-C-S-G-P-A-D-T-G-R; (SEQ ID NO: 8)Y-M-A-N-G-C-L-sL-N-Y-L-R; (SEQ ID NO: 9)I-C-D-F-G-T-A-C-D-I-Q-T-H-M-T-N-N-K; and (SEQ ID NO: 10)Y-F-S-N-R-P-G-P-T-P-G-C-Q-L-P-(13C6-15N4)R-P-N-C- P-V-E-T-L-K.

In some embodiments, the polypeptide is a kinase fragment comprisingfrom about 10 to about 30 amino acid residues. In some embodiments, atleast one of the polypeptides comprises an amino acid sequence having atleast 90% sequence identity to a sequence selected from the groupconsisting of:

(SEQ ID NO: 1) L-M-D-A-N-L-C-Q-V-I-Q-M-E; (SEQ ID NO: 2)L-V-M-E-Y-L-P-S-G-C-L-R; (SEQ ID NO: 3)M-A-P-P-D-L-P-H-W-Q-D-C-H-E-L-W-S-K; (SEQ ID NO: 4)H-G-C-L-S-D-Y-L-R-S-Q-R-G-L-F-A-A-E; (SEQ ID NO: 5)Y-F-S-N-R-P-G-P-T-P-G-C-Q-L-P-R-P-N-C-P-V-E-T-L-K; (SEQ ID NO: 6)G-C-L-L-D-Y-V-R; (SEQ ID NO: 7) F-G-L-C-S-G-P-A-D-T-G-R; and(SEQ ID NO: 8) Y-M-A-N-G-C-L-sL-N-Y-L-R.

In some embodiments, the method further comprises reacting one or moretest compounds, each containing a thiol-reactive moiety, with a secondcompound containing a nucleophilic thiol group to form one or more testcompound thioether conjugates, prior to the contacting of step i). Insome embodiments, the second compound is selected from the groupconsisting of beta-mercaptoethanol, glutathione, 2-mercaptobenzoic acid,and hydrogen sulfide.

In some embodiments, the method further comprises reacting one or moreacrylamide compounds with a thiolated compound to form one or moreacrylamide thiolated derivatives, prior to the contacting of step i).

In some embodiments, the method further comprises analyzing the one ormore test compound thioether conjugates in a mass spectrometry assayprior to the contacting step i). In some embodiments, analyzing the testcompound thioether conjugates comprises generating a database offragment ion spectra comprising the mass spectra of each of the one ormore test compound thioether conjugates. In some embodiments, the methodfurther comprises gas-phase isolation and fragmentation (e.g., MS/MS/MSor MS3) of the one or more of the thiolated ions, or derivative ionsthereof, formed during MS/MS of the one or more test compound thioetherconjugates.

In some embodiments, the method further comprises analyzing the one ormore acrylamide thiolated derivatives in a mass spectrometry assay. Insome embodiments, analyzing the one or more acrylamide thiolatedderivatives comprises generating a database of fragment ion spectracomprising the mass spectra of each of the one or more acrylamidethiolated derivatives.

In some embodiments, the method further comprises isolating the one ormore thiolated ions after the detecting of step iii). In someembodiments, the method further comprises gas-phase isolation andfragmentation (e.g., MS/MS/MS or MS3) of the one or more thiolated ions,or derivative ions thereof, detected in step iii).

In some embodiments, the method further comprises performing a massspectrometry assay on the one or more isolated thiolated ions prior tothe identifying of step iv).

In some embodiments, the identifying of step iv) further comprisesidentifying a mass spectrum in the database of fragment ion spectra(e.g., MS/MS/MS or MS3 fragment ion spectra) from the one or more testcompound thioether conjugates that is substantially identical to thefragment ion spectrum derived from the gas-phase isolation andfragmentation analysis (e.g., MS/MS/MS or MS3) performed on thethiolated ion detected in step iii).

In some embodiments, the identifying of step iv) further comprisesidentifying a mass spectrum in the database of fragment ion spectra thatis substantially identical to the mass spectrum of the isolatedthiolated ion. In some embodiments, the thiolated compound isβ-mercaptoethanol.

In some embodiments, the first mixture comprises more than one testcompound. In some embodiments the first mixture comprises a combinationof one or more test compounds and one or more chemically-modified testcompounds (e.g., a test compound comprising a peptide-epitope, biotin,desthiobiotin, or a bio-orthogonal moiety such as an alkyne or azide).In some embodiments, the second mixture comprises more than onepolypeptide. In some embodiments, the first mixture comprises more thanone test compound and the second mixture comprises more than onepolypeptide. In some embodiments the first mixture comprises acombination of one or more test compounds and one or morechemically-modified test compounds (e.g., a test compound comprising apeptide-epitope, biotin, desthiobiotin, or a bio-orthogonal moiety suchas an alkyne or azide), and the second mixture comprises more than onepolypeptide.

The present application further provides an analytical method,comprising:

-   -   i) reacting one or more test compounds each containing a        thiol-reactive moiety with a second compound containing a thiol        group to form one or more test compound thioether conjugates;    -   ii) analyzing the one or more test compound thioether conjugates        in a mass spectrometry assay;    -   iii) generating a database of fragment ion spectra comprising        the mass spectra derived from the mass spectrometry assay (e.g.,        MS/MS/MS or MS3) performed on each of the one or more test        compound thioether conjugates;    -   iv) contacting a first mixture comprising more than one test        compound with a second mixture comprising more than one        polypeptide to form a third mixture comprising more than one        test compound-polypeptide conjugate, wherein each of the test        compound-polypeptide conjugates comprise one or more thioether        bonds;    -   v) analyzing the third mixture using a mass spectrometry assay;    -   vi) detecting one or more thiolated ions, or derivative ions        thereof, produced in the mass spectrometry assay;    -   vii) performing gas phase isolation and MS/MS/MS or MS3 analysis        on the one or more thiolated ions, or derivative ions thereof,        to generate fragment ion spectra;    -   viii) comparing the fragment ion spectra generated in step vii)        with the database of fragment ion spectra generated in step        iii); and    -   ix) identifying that one or more of the test compounds binds        irreversibly to one or more of the polypeptides based on the        detection of one or more thiolated ions, or derivative ions        thereof, in the mass spectrometry assay of step v) and the        identification of a mass spectrum in the database of fragment        ion spectra that is substantially identical to the mass spectrum        of the thiolated ion, or derivative ion thereof, generated in        step iii).

In some embodiments, the fragment ion spectra generated in step vii) arediagnostic of the one or more test compounds.

The present application further provides an analytical method,comprising:

-   -   i) reacting one or more acrylamide compounds with a        thiol-containing compound to form one or more acrylamide        thiolated derivatives;    -   ii) analyzing the one or more acrylamide thiolated derivatives        in a mass spectrometry assay;    -   iii) generating a database of fragment ion spectra comprising        the mass spectra of each of the one or more acrylamide thiolated        derivatives;    -   iv) contacting a first mixture comprising more than one test        compound with a second mixture comprising more than one        polypeptide to form a third mixture comprising more than one        compound-polypeptide conjugate, wherein each of the        compound-polypeptide conjugates comprise one or more thioether        bonds;    -   v) analyzing the third mixture using a mass spectrometry assay;    -   vi) detecting one or more thiolated ions produced in the mass        spectrometry assay;    -   vii) isolating the one or more thiolated ions;    -   viii) performing a mass spectrometry assay on the one or more        isolated thiolated ions;    -   ix) comparing the mass spectra of the one or more thiolated ions        to the database of fragment ion spectra; and    -   x) identifying that one or more of the test compounds binds        irreversibly to one or more of the polypeptides based on the        detection of one or more thiolated ions in the mass spectrometry        assay of step v) and the identification a mass spectrum in the        database of fragment ion spectra that is substantially identical        to the mass spectrum of the isolated thiolated ion of step        viii).

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Methods and materials aredescribed herein and in Appendix A of U.S. Provisional PatentApplication No. 62/427,042 (the disclosure of which is incorporatedherein by reference in its entirety) for use in the present invention;other, suitable methods and materials known in the art can also be used.The materials, methods, and examples are illustrative only and notintended to be limiting. All publications, patent applications, patents,sequences, database entries, and other references mentioned herein areincorporated by reference in their entirety. In case of conflict, thepresent specification, including definitions, will control.

DESCRIPTION OF DRAWINGS

FIGS. 1A-1C show acrylamide kinase inhibitor/target conjugates generatepredictable thiolated ions. MS/MS spectra for (FIG. 1A) JNK-IN-2 labeledJNK1 peptide; (FIG. 1B) Ibrutinib labeled ITK peptide; and (FIG. 1C)Neratinib labeled EGFR peptide. Fragment ions containing the peptide N-(b-type) or C- (y-type) termini are indicated with glyphs above andbelow the peptide sequence. Modified cysteine residues are shown inbold, italic font. Thiolated ions are denoted with ‘*’. Structures forthiolated ions are shown adjacent to each mass spectrum.

FIGS. 2A-2D show pre-processing peak lists from fragment ion spectra toaccount for inhibitor-related dissociation pathways, which significantlyincreases MASCOT peptide scores. (FIG. 2A) The deisotoped MS/MS spectrumfor a THZ531 labeled CDK12 peptide yields a relatively low-confidenceMASCOT score, largely due to an abundance of inhibitor related fragmentions (FIG. 2B, #1-#9; see Table 3 for proposed structures). Additionalsteps, comprising normalization of all fragment ions to z=1, includingneutral loss of inhibitor as part of MASCOT variable-mod definition, andsubtraction of peaks corresponding to internal fragmentation of theinhibitor yields a high-confidence MASCOT peptide score (FIG. 2C). Ionsof type b and y are indicated with glyphs above and below the peptidesequence. Neutral loss ions are indicated with open circles. ++indicates a doubly charged ion. (FIG. 2D) Scatter plots of MASCOT scoresfor BSA peptides modified by (left) THZ531 or (right) THZ1 for MS/MSdata subject to deisotoping (y-axis) or full spectral preprocessing(x-axis). Dotted lines represent MASCOT score cutoffs for a 1% FDR.

FIGS. 3A-3C show normalized fragment ion intensity vs. collision energy(CE) for inhibitor specific (thiolated and iy1) ions produced by MS/MSof a triply charged synthetic cysteine-containing peptide (FGLCSGPADTGR(SEQ ID NO: 7)) labeled with (FIG. 3A) Ibrutinib; (FIG. 3B) Neratinib;and (FIG. 3C) QL47. For comparison, each plot includes profiles forb-/y-type ions produced by MS/MS of the unlabeled triply chargedpeptide.

FIGS. 4A-4G show novel dissociation pathways associated with thioetherlinked covalent probes, which provide for significant gas-phaseenrichment during precursor ion scanning mass spectrometry. (FIG. 4A)Base-peak chromatogram (BPC) and (FIG. 4B) extracted ion chromatogram(XIC) from Q3MS scans and (FIG. 4C) individual Q3 full-scan massspectrum recorded during analysis of a synthetic cysteine-containingpeptide (FGLCSGPADTGR (SEQ ID NO: 7); indicated by “CYS”) labeled withIbrutinib and spiked into a mix of tryptic peptides derived from humanmyeloid K562 cells. Arrow indicates the elution time (FIGS. 4A and 4B)or m/z (FIG. 4C) for the labeled peptide. (FIG. 4D) Base-peakchromatogram, and (FIG. 4E) extracted ion chromatogram from precursorion spectra (precursors of 475.19, corresponding to the thiolated ion ofIbrutinib; abbreviated as “Prec”), and (FIG. 4F) individual precursorion mass spectrum recorded during the same LC-MS/MS analysis. Arrowindicates the elution time (FIGS. 4D and 4E) or precursor ion signal(FIG. 4F) for the Ibrutinib labeled peptide. (FIGS. 4C and 4F) The %values in each panel represent the gas phase enrichment, calculated asthe relative contribution of each ion (arrow) as compared to the totalion current in that spectrum. (FIG. 4G) MS/MS spectrum of Ibrutiniblabeled peptide triggered by precursor scans for m/z=475.19,corresponding to the thiolated ion of Ibrutinib. Fragment ionscontaining the peptide N- (b-type) or C- (y-type) termini are indicatedwith glyphs above and below the peptide sequence. Inhibitor-specificions are labeled with numbers (see Table 3 for proposed structures ofions labeled #1-5).

FIG. 5 shows collision energy profiles for peptideF-G-L-C-S-G-P-A-D-T-G-R (SEQ ID NO.: 7; left column) and peptideY-F-S-N-R-P-G-P-T-P-G-C-Q-L-P-(¹³C₆-¹⁵N₄)-R-P-N-C-P-V-E-T-L-K (SEQ IDNO.: 10; right column) alkylated with 15 broad thiol-reactive reagents.The intensities of peptide-derived y3 and a2 ions (for peptidesF-G-L-C-S-G-P-A-D-T-G-R (SEQ ID NO.: 7) andY-F-S-N-R-P-G-P-T-P-G-C-Q-L-P-(¹³C₆-¹⁵N₄)-R-P-N-C-P-V-E-T-L-K (SEQ IDNO.: 10, respectively) and reagent-derived ions (see e.g., Table 4) areplotted as a function of collision energy (in eV).

FIG. 6 shows CDK7 target engagement (TE) quantification. CDK7 wasimmunopurified from DMSO and THZ1-treated cells. Immunopurified CDK7were labeled with light DMPIA (THZ1-treated) and heavy DMPIA(DMSO-treated) before trypsin digestion, as described in Example 8.eXtracted Ion Chromatograms (XIC) for light DMPIA and heavyDMPIA-derived fragment ions (m/z: 160.08 and m/z: 162.08 respectively)are shown.

FIG. 7 shows a representative diagram visualizing the workflow of thetarget engagement stoichiometry assay described in Example 8.

FIGS. 8A-8H show common fragmentation pathways observed in MS/MS spectraof inhibitor conjugated peptides. MS/MS spectra for inhibitor targetconjugates (FIG. 8A) THZ531 labeled CDK12 peptide; (FIG. 8B) TL10-201labeled JAK3 peptide; (FIG. 8C) THZ1 labeled CDK7 peptide; (FIG. 8D)HBX-19818 labeled USP7 peptide. MS/MS spectra of the syntheticcysteine-containing peptide FGLCSGPADTGR (SEQ ID NO: 7) conjugated to(FIG. 8E) QL-47; (FIGS. 8F, 8G) HBX-19818; and (FIG. 8H) MI-2. Fragmentions containing the peptide N- (b-type) or C- (y-type) termini areindicated with glyphs above and below the peptide sequence. Modifiedcysteine residues are shown in bold, italic print. Thiolated ions aredenoted with ‘*’. (FIG. 8F) At a CE of 20 eV HBX-19818 modified peptideeliminates the inhibitor/thiol to produce an intense thiolated ion(marked ‘*’) and a series of dehydroalanine containing b-/y-type ions(marked with open circles) along with b-/y-type ions (marked by filledcircles). (FIG. 8G) At higher CE (65 eV) the HBX-19818 modifiedsynthetic peptide produces an ib₁ ion (1) and other structure-specificions (2, 3). Imm(F) indicates the immonium ion of phenylalanine. (FIG.8H) MI-2 modified peptide also shows a peak corresponding to amide bondcleavage (“1”).

FIGS. 9A-9B show bar graphs illustrating shift in charge statedistribution observed after conjugation of reduced BSA peptides to (FIG.9A) THZ1 (CDK7 inhibitor) or (FIG. 9B) THZ531 (CDK12 inhibitor) comparedto the same peptides alkylated with iodoacetamide.

FIGS. 10A-10F show normalized fragment ion intensity vs. collisionenergy (CE) for inhibitor specific (thiolated and iy1) ions produced byMS/MS of triply charged synthetic cysteine-containing peptideFGLCSGPADTGR (SEQ ID NO: 7) labeled with (FIG. 10A) TL10-201; (FIG. 10B)THZ1; (FIG. 10C) THZ531. For comparison, each plot includes profiles forb-/y-type ions produced by MS/MS of the unlabeled triply chargedpeptide. (FIG. 10D) Signal intensity as a function of collision energyfor inhibitor-specific fragments recorded during MS/MS analysis oftriply charged BTK peptide conjugated to JNK-IN-2 (JNK1 inhibitor).(FIGS. 10E and 10F) Signal intensity as a function of collision energyfor thiolated ions recorded during MS/MS analysis of doubly- ortriply-charged FGLCSGPADTGR (SEQ ID NO: 7) or BTK peptides conjugated to(FIG. 10E) TL10-201 (JAK3 inhibitor) or (FIG. 10F) JNK-IN-2 (JNKinhibitor).

FIGS. 11A-11F show that a second dissociation pathway can be used toafford selective detection of an Ibrutinib labeled peptide by precursorion mass spectrometry. (FIG. 11A) Base-peak chromatogram (BPC) and (FIG.11B) extracted ion chromatogram (XIC) from Q3MS scans and (FIG. 11C)individual Q3 full-scan mass spectrum recorded during analysis of asynthetic cysteine-containing peptide labeled with Ibrutinib(FGLCSGPADTGR (SEQ ID NO: 7); indicated by CYS) and spiked into a mix oftryptic peptides derived from human myeloid K562 cells. Arrow indicatesthe elution time (FIGS. 11A and 11B) or m/z (FIG. 11C) for the labeledpeptide. These data were generated from the same LC-MS/MS analysis as inFIGS. 4A-4G. Base-peak chromatogram and (FIG. 11E) extracted ionchromatogram from precursor ion spectra (precursors of 304.12,corresponding to alkylated amine cleavage; abbreviated as “Prec”), andindividual precursor ion mass spectrum (FIG. 11F) recorded during thesame LC-MS/MS analysis. Arrow indicates the elution time (FIGS. 11D and11E) or precursor ion signal (FIG. 11F) for the Ibrutinib labeledpeptide. (FIGS. 11C and 11F) The % values in each panel represent thegas phase enrichment, calculated as the relative contribution of eachion (arrow) as compared to the total ion current in that spectrum.

FIGS. 12A-12J show selective detection of QL47-modified peptides usingprecursor ion scanning mass spectrometry. (FIG. 12A) Base-peakchromatogram (BPC) and (FIG. 12B) extracted ion chromatogram (XIC) fromQ3MS scans and (FIG. 12C) individual Q3 full-scan mass spectrum recordedduring analysis of a synthetic cysteine-containing peptide labeled withQL47 (FGLCSGPADTGR (SEQ ID NO: 7); indicated by CYS) and spiked into amix of tryptic peptides derived from human myeloid K562 cells. Arrowindicates the elution time (FIGS. 12A-12B) or m/z (FIG. 12C) for thelabeled peptide. (FIGS. 12D and 12G) Base-peak chromatograms; (FIGS. 12Eand 12H) extracted ion chromatograms from precursor ion spectra; and(FIGS. 12F and 12I) individual precursor ion mass spectra recordedduring the same LC-MS/MS analysis (FIGS. 12D-12F, precursors of 482.16,corresponding to the thiolated ion of QL47; FIGS. 12G-12I, precursors of394.16, corresponding to the iy₁ ion of QL47; abbreviated as “Prec”).Arrow indicates the elution time (FIGS. 12D, 12E, 12G, and 12H) orprecursor ion signal (FIGS. 12F and 12I) for the labeled peptide. (FIGS.12C, 12F, and 12I) The % values in each panel represent the gas phaseenrichment, calculated as the relative contribution of each ion (arrow)as compared to the total ion current in that spectrum. (FIG. 12J)Proposed structures and calculated masses of the QL47 thiolated (left)and iy₁ (right) ions.

FIGS. 13A-13B illustrate creation of a spectral library of test compoundderived thiolated ions. In FIG. 13A, each test compound is reacted withβ-mercaptoethanol or other nucleophilic thiol reagent (e.g.,glutathione, 2-mercaptobenzoic acid, hydrogen sulfide, etc.) to form aninactivated test compound. In some embodiments, chemically-modified testcompounds are treated with desthiobiotin (DTB)-PEG3-azide in thepresence of copper, TCEP, and ligand (for example TBTA) to promotecopper catalyzed azide-alkyne cycloaddition (CuCAAC) resulting in theattachment of a desthiobiotin affinity tag. MS/MS of the inactivatedtest compound will generate test compound-specific thiolated ions in thegas phase. MS/MS/MS performed on these thiolated ions generatesreference spectra for thiolated ions derived from each test compound.Reference spectra are then assembled into a spectral library databaseusing standard software tools such as those available from the NationalInstitute of Standards and Technology (NIST) (chemical structures areprovided for illustrative purposes). In FIG. 13B, eachchemically-modified test compound is reacted with β-mercaptoethanol orother nucleophilic thiol reagent (e.g., glutathione, 2-mercaptobenzoicacid, hydrogen sulfide, etc.) to form an inactivated,chemically-modified test compound. For test compounds which arechemically-modified to contain an alkyne moiety, one of severalbio-orthogonal strategies, such as click-chemistry or the Staudingerligation, may be used to incorporate an affinity tag (e.g., adesthiobiotin affinity tag). MS/MS of the chemically-modified,inactivated test compound will generate test compound-specific thiolatedions in the gas phase. MS/MS/MS performed on these thiolated ionsgenerates reference spectra for thiolated ions derived from eachchemically-modified test compound. Reference spectra are then assembledinto a spectral library database using standard software tools such asthose available from the National Institute of Standards and Technology(NIST) (chemical structures are provided for illustrative purposes).

FIGS. 14A-14B show sample processing workflows for massively multiplexedChemoformics assay. In FIG. 14A, individual cell cultures are firstincubated with increasing concentrations of test compound. After removalof excess test compound, cells are lysed and proteins digested withtrypsin or other endoprotease. Resulting peptides are labeled with TMTor other multiplexed stable isotope reagents. Mass spectrometry dataacquisition is performed as described in FIG. 15 (chemical structuresare for illustrative purposes). In FIG. 14B, individual cell culturesare first incubated with increasing concentrations of test compound asshown in FIG. 14A. Next, individual cell cultures are incubated with afixed concentration of a chemically-modified analog of each of thenative (i.e., not chemically-modified) test compounds used in the firstincubation step (native and chemically-modified test compounds arerepresented as different colored shapes). Excess test andchemically-modified test compounds are removed, and cells are lysed. Fortest compounds which are chemically-modified to contain an alkynemoiety, one of several bio-orthogonal strategies, such asclick-chemistry or the Staudinger ligation, may be used to incorporatean affinity tag (e.g., a desthiobiotin affinity tag). In someembodiments, protein lysates are treated with desthiobiotin(DTB)-PEG3-azide in the presence of copper, TCEP, and ligand (forexample TBTA) to promote copper catalyzed azide-alkyne cycloaddition(CuCAAC) resulting in the attachment of a desthiobiotin affinity tag toevery protein irreversibly bound by a chemically-modified test compound.Desthiobiotin-tagged proteins can be enriched by use of avidin orstreptavidin beads and the enriched set of proteins is digested withtrypsin or other endoprotease. The digestion step may be performedeither on bead-bound proteins or after elution of proteins from theavidin or streptavidin beads. The resulting peptides are labeled withTMT or other multiplexed stable isotope reagents. Mass spectrometry dataacquisition is performed as described in FIG. 15 . Alternatively, aftersteps 1-5, proteins can be digested and DTB tagged peptides enrichedusing avidin or streptavidin beads. After elution, peptides are labeledwith TMT or other multiplexed stable isotope reagents and massspectrometry data acquisition is performed as described in FIG. 15 .

FIG. 15 shows a custom data acquisition scheme for Chemoformics assay. Ahigh resolution MS1 scan records m/z values of tryptic peptides, some ofwhich will be covalently modified by test compounds. Small segments ofm/z space (e.g., ˜20 Da) are incrementally subjected to MS/MS andspectra screened for thiolated ions or derivative ions thereof. Whensuch ions are detected, MS/MS/MS is used to fragment the thiolated ionand the resulting spectrum is searched against a previously generatedthiolated ion spectral library (see e.g., FIG. 13 ). If there is a matchin the spectral database, high resolution MS/MS scans are acquiredwithin the original m/z window (e.g., ˜20 Da). MS/MS spectra are matchedto parent protein sequences using MASCOT, with variable modification ofcysteine set according to the inhibitor identified by the spectrallibrary search. The cycle is repeated until the end of the LC gradient.

FIG. 16 shows features of a hypothetical representative ChemoformicActivity Map. Proteins detected based on cysteine residues modified bytest compounds are listed on the y-axis. Each test compound used in theChemoformic assay is listed on the x-axis. Color gradients indicatedose-dependent covalent binding for test compounds at differentprotein-cysteine residues. Some test compounds may covalently modifymultiple proteins in a given family such as kinases, deubiquitinatingenzymes, etc. These protein families may cluster within the ChemoformicActivity Map (e.g., “A”, “B”, “C” of FIG. 16 ). Non-selective covalenttest compounds (e.g. “promiscuous probes” of FIG. 16 ) may exhibithighly promiscuous activity and covalently bind large numbers ofproteins; similarly, a subset of cysteine residues on a small number ofproteins may be highly reactive and form covalent bonds with a largenumber of test compounds (e.g., “promiscuous proteins” of FIG. 16 ).

DETAILED DESCRIPTION

Selective covalent inhibitors which utilize diverse reactive ‘warheads’have been developed for numerous enzyme families. A subset of these hasbeen successfully produced to yield clinical-grade probes targetingkinases deubiquitinating enzymes and other catalytically activeproteins. Despite these promising results the characterization ofon-/off-target molecules for lead compounds, in addition to subsequentmedicinal chemistry optimization remains a significant challenge. Massspectrometry is an integral component of analytical platforms used tocharacterize covalent probes. Several approaches have been developedthat rely on direct detection of targets based on affinity-tagged probesor the use of broad-reactivity reagents in a competition format withnative inhibitors to provide an indirect readout of targets. Althoughinformative, these approaches may be limited by (i) the use of taggedanalogues which may not faithfully reproduce the physicochemicalproperties of the native probe or (ii) stochastic properties of shotgunLC-MS/MS whereby low-expression targets or those labeled atlow-stoichiometry are not reproducibly detected or quantified.

The present application describes that cysteine side chains covalentlymodified via a thioether linkage exhibit common gas-phase fragmentationpathways. As described herein, these inhibitor-specific fragment ionshave been leveraged to (i) significantly improve identification ofmodified peptides via commercial search algorithms and, (ii) facilitateselective detection of inhibitor-modified peptides in complex mixtures.

The fragmentation behavior is specific to each probe. As a result, a setor library of probes provides a means to chemically encode targetproteins or other biomolecules. The combination of this chemicallyencoded diversity with isobaric stable isotope labeling provides, forexample, a means to achieve multiplexing for discovery experimentswhereby mass spectrometry is used to characterize proteins or otherbiomolecules irreversibly bound by covalent probes.

In addition, the gas phase fragmentation behavior specific to each probecan be used to develop targeted mass spectrometry assays to provide ahigh-throughput readout of target engagement stoichiometry. These assayshave applicability in drug discovery, for example, biochemicalcharacterization of lead compounds, use in pre-clinical models, clinicaltrials, and as standard clinical assays in point-of-care settings.

Accordingly, the present application provides, inter alia, analyticalmethod for identifying whether a test compound irreversibly bonds to apolypeptide based on the detection of one or more thiolated or otherinhibitor-specific fragment ions in a mass spectrometry assay.

In some embodiments, the analytical method comprises:

-   -   i) contacting or reacting a test compound with a polypeptide to        form a test compound-polypeptide conjugate;    -   ii) analyzing the test compound-polypeptide conjugate using a        mass spectrometry assay;    -   iii) detecting thiolated or other inhibitor-specific ions (e.g.,        derivative ions thereof described herein) produced in the mass        spectrometry assay; and    -   iv) identifying that the test compound irreversibly bonds to the        polypeptide based on the detection of the thiolated or other        inhibitor-specific ions (e.g., derivative ions thereof described        herein) in the mass spectrometry assay.

As used herein, the term “thiolated ions” refers to one or more ionsformed in the mass spectrometry assays described herein corresponding tocleavage of a polypeptide-test compound conjugate, wherein the thiolatedion corresponds to a thiol-derivative of the test compound (e.g., athiol-derivative of the kinase inhibitor or a thiol-derivative of thedeubiquitinase inhibitor).

As used herein, the term “derivative ions” refers to fragment ions ofthe test compound-polypeptide conjugates that are formed during the massspectrometry assays described in the methods provided herein. Exemplary“derivative ions” may be formed, for example, due to variousintramolecular elimination reactions such as the cleavage of amide bondswithin a test compound.

As used herein, the term “acrylamide-thiolated derivatives” refers to anacrylamide compound (e.g., an acrylamide test compound) which has beenreacted with a thiol containing reagent to form an inactivated thioetherconjugate (e.g., a test compound-thioether conjugate).

In some embodiments, the present application provides analytical methodsfor identifying whether a test compound irreversibly bonds to apolypeptide based on the detection of one or more thiolated ions in amass spectrometry assay.

In some embodiments, the analytical method comprises:

-   -   i) contacting a test compound with a polypeptide to form a test        compound-polypeptide conjugate;    -   ii) analyzing the test compound-polypeptide conjugate using a        mass spectrometry assay;    -   iii) detecting one or more thiolated ions produced in the mass        spectrometry assay; and    -   iv) identifying that the test compound irreversibly bonds to the        polypeptide based on the detection of the one or more thiolated        ions in the mass spectrometry assay.

In some embodiments, the compound-polypeptide conjugate comprises one ormore thioether bonds between the test compound and the polypeptide. Insome embodiments, the compound-polypeptide conjugate comprises onethioether bond between the test compound and the polypeptide. In someembodiments, the compound-polypeptide conjugate comprises more than one(e.g., two, three, or four) thioether bond between the test compound andthe polypeptide.

In some embodiments, the irreversible bond is an irreversible covalentbond.

In some embodiments, step i) comprises contacting the test compound andthe polypeptide in the presence of a first solvent component. In someembodiments, the first solvent component comprises one or more aproticsolvents. In some embodiments, the first solvent component comprises asingle solvent. In some embodiments, the first solvent componentcomprises a single aprotic solvent. In some embodiments, the firstsolvent component is DMSO.

In some embodiments, step i) comprises treating cells growing in culturemedia with the test compound. In some embodiments, the culture media isRPMI-1640. In some embodiments, the culture media is DMEM-F12. In someembodiments, the culture media further comprises FBS.

In some embodiments, step i) comprises treating cell lysates with thetest compound. In some embodiments, the lysates are prepared with NP-40.In some embodiments, lysates are prepared with Triton X-100.

In some embodiments, step i) further comprises contacting the compoundand the polypeptide in the presence of a buffer agent. Example bufferagents include, but are not limited to, carbonate buffer agents,bicarbonate buffer agents, phosphate buffer agents, citric acid/citratebuffer agents, ammonium formate,3-[[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino]propane-1-sulfonicacid (TAPS), bicine, 2-amino-2-hydroxymethyl-propane-1,3-diol (Tris),tricine,3-[[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino]-2-hydroxypropane-1-sulfonicacid (TAPSO), 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid(HEPES),2-[[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino]ethanesulfonic acid(TES), 3-Morpholinopropane-1-sulfonic acid (MOPS),1,4-Piperazinediethanesulfonic acid (PIPES), and2-morpholin-4-ylethanesulfonic acid (2-morpholin-4-ylethanesulfonicacid). In some embodiments, the buffer agent is triethylammoniumbicarbonate. In some embodiments, the buffer agent comprises TRIS andammonium format.

In some embodiments, step i) is performed at a temperature of about 4°C. to about 100° C., for example, about 4° C. to about 100° C., about 4°C. to about 80° C., about 4° C. to about 40° C., about 4° C. to about30° C., about 4° C. to about 20° C., about 20° C. to about 100° C.,about 20° C. to about 80° C., about 20° C. to about 60° C., about 20° C.to about 40° C., about 20° C. to about 30° C., about 30° C. to about100° C., about 30° C. to about 80° C., about 30° C. to about 60° C.,about 30° C. to about 40° C., about 40° C. to about 100° C., about 40°C. to about 80° C., about 40° C. to about 60° C., about 60° C. to about100° C., about 60° C. to about 80° C., or about 80° C. to about 100° C.In some embodiments, step i) is performed at a temperature of about 30°C. to about 65° C. In some embodiments, step i) is performed at atemperature of about room temperature. In some embodiments, step i) isperformed at a temperature below room temperature.

In some embodiments, step i) is performed for about 1 minute to about 48hours, for example, from about 1 minute to about 48 hours, about 10minute to about 24 hours, about 1 minute to about 18 hours, about 1minute to about 16 hours, about 1 minute to about 12 hours, about 1minute to about 8 hours, about 1 minute to about 1 hours, about 1 hourto about 18 hours, about 1 hour to about 16 hours, about 1 hour to about12 hours, about 1 hour to about 8 hours, about 8 hours to about 24hours, about 8 hours to about 18 hours, about 8 hours to about 16 hours,about 8 hours to about 12 hours, about 12 hours to about 24 hours, about12 hours to about 18 hours, about 12 hours to about 16 hours, about 16hours to about 24 hours, about 16 hours to about 18 hours, or about 18hours to about 24 hours. In some embodiments, step i) is performed forabout 8 hours to about 16 hours.

In some embodiments, step i) is performed using a molar excess of thetest compound compared to the polypeptide. In some embodiments, themolar ratio of the test compound to the polypeptide is from about 0.1:1to about 100:1, for example, from about 0.1:1 to about 100:1, from about0.1:1 to about 50:1, from about 0.1:1 to about 25:1, from about 0.1:1 toabout 20:1, from about 0.1:1 to about 15:1, from about 0.1:1 to about10:1, from about 0.1:1 to about 2:1, from about 0.1:1 to about 1:1, fromabout 1:1 to about 100:1, from about 1:1 to about 50:1, from about 1:1to about 25:1, from about 1:1 to about 20:1, from about 1:1 to about15:1, from about 1:1 to about 10:1, from about 1:1 to about 2:1, fromabout 2:1 to about 100:1, from about 2:1 to about 50:1, from about 2:1to about 25:1, from about 2:1 to about 20:1, from about 2:1 to about15:1, from about 2:1 to about 10:1, from about 10:1 to about 100:1, fromabout 10:1 to about 50:1, from 10:1 to about 25:1, from about 10:1 toabout 20:1, from about 10:1 to about 15:1, from about 15:1 to about100:1, from about 15:1 to about 50:1, from 15:1 to about 25:1, fromabout 15:1 to about 20:1, from about 20:1 to about 100:1, from about20:1 to about 50:1, from 20:1 to about 25:1, from about 25:1 to about100:1, from about 25:1 to about 50:1, or from about 50:1 to about 100:1.In some embodiments, the molar ratio of the test compound to thepolypeptide is from about 5:1 to about 15:1.

In some embodiments, the methods provided herein further comprisepreparing the test compound-polypeptide conjugate for mass spectrometryanalysis. For example, this preparation may involve one or more steps ofchromatography (e.g., ion exchange, reversed phase) and exchange into asuitable solvent. In some embodiments, the reaction mixture may bediluted in a suitable solvent (e.g., 30% acetonitrile with 0.1% aceticacid) and injected directly into the mass spectrometer.

In some embodiments, the method provided herein further comprisescontacting the test compound-polypeptide conjugate with an acid in thepresence of a second solvent component prior to performing the massspectrometry assay of step ii). Example acids can be inorganic ororganic acids and include, but are not limited to, strong and weakacids. Some strong acids include hydrochloric acid, hydrobromic acid,sulfuric acid, phosphoric acid, p-toluenesulfonic acid, 4-nitrobenzoicacid, methanesulfonic acid, benzenesulfonic acid, trifluoroacetic acid,and nitric acid. Some weak acids include, but are not limited to, aceticacid, propionic acid, butanoic acid, benzoic acid, tartaric acid,pentanoic acid, hexanoic acid, heptanoic acid, octanoic acid, nonanoicacid, and decanoic acid. In some embodiments, the acid is an organicacid. In some embodiments, the acid is acetic acid.

In some embodiments, the second solvent component comprises one or moreaprotic solvents. In some embodiments, the second solvent componentcomprises a single solvent. In some embodiments, the second solventcomponent comprises a single aprotic solvent. In some embodiments, thesecond solvent component comprises acetonitrile. In some embodiments,the second solvent component further comprises one or more proticsolvents. In some embodiments, the second solvent component furthercomprises water.

In some embodiments, the method provided herein further comprisesdigesting the test compound-polypeptide conjugate prior to theperforming the mass spectrometry assay of step ii). In some embodiments,the resulting compound-peptide conjugates are useful for traditionalshotgun and/or targeted mass spectrometry assays (e.g., MRM, SRM,precursor ion, and the like).

In some embodiments, the digesting is performed in the presence of adigestive enzyme. In some embodiments, the digesting comprises reactingthe test compound-polypeptide conjugate with trypsin or anotherproteolytic enzyme in the presence of a third solvent component.

In some embodiments, the third solvent component comprises ammoniumbicarbonate. In some embodiments, the third solvent component comprisesaqueous ammonium bicarbonate. In some embodiments, the third solventcomponent comprises 100 mM ammonium bicarbonate in water.

In some embodiments, proteins from cells and/or lysates are prepared formass spectrometry analysis as described herein. For example, detergentsare removed before or after digestion and proteins are reduced andalkylated. In some embodiments, proteins and/or lysates may be treatedwith one or more chaotropic agents (e.g., Urea/GuHCl). In someembodiments, the proteins and/or lysates may be digested with aproteolytic enzyme (e.g., trypsin, GluC, AspN, pepsin, trypN, elastase,ArgC, and chymotrypsin). In some embodiments, the proteins and/orlysates may be chemically digested (e.g., with cyanogen bromide orhydroxylamine). In some embodiments, the proteins and/or lysates may bedesalted (e.g., by reversed phase). In some embodiments, the detergentmay be removed by acetone precipitation, trizol extraction, ion exchangechromatography, or other solid phase extraction techniques commonly usedin the field.

In some embodiments, the polypeptide comprises one or more amino acidsresidues comprising at least one sulfur atom. In some embodiments, thepolypeptide comprises one or more cysteine residues. In someembodiments, the polypeptide comprises one cysteine residue. In someembodiments, the polypeptide comprises more than one cysteine residue.

In some embodiments, the test compound comprises one or more functionalgroups comprising at least one sulfur atom. In some embodiments, thetest compound comprises one or more cysteine groups. In someembodiments, the test compound comprises one cysteine group. In someembodiments, the test compound comprises more than one cysteine group.In some embodiments the test compound may contain one or more aminoacids or linked amino acids in addition to one or more functional groupscontaining at least one sulfur atom.

In some embodiments, the test compound is capable of irreversiblybonding to at least one of the one or more amino acids residuescomprising at least one sulfur atom in the polypeptide. In someembodiments, the test compound is capable of irreversibly bonding to oneor more cysteine residues of the polypeptide. In some embodiments, thetest compound is capable of irreversibly bonding to one of the cysteineresidues of the polypeptide. In some embodiments, the test compound iscapable of irreversibly bonding to more than one of the cysteineresidues of the polypeptide.

In some embodiments, the test compound comprises one or more groupscapable of forming thioether bonds with the polypeptide (e.g., with oneor more cysteine residues in the polypeptide). In some embodiments, thetest compound comprises one or more acrylamide groups (e.g., asubstituted or unsubstituted acrylamide group). In some embodiments, thetest compound comprises one or more acrylamide groups, dimethylaminoacrylamide groups, iodoacetamide groups, chloroacetamide groups,maleimide groups, or reactive C—X groups, wherein X is a halogen. Insome embodiments, the test compound is isotopically labeled with heavyisotopes of carbon, oxygen, nitrogen, sulfur, phosphorous, chlorine,bromine, or hydrogen. In some embodiments, the test compound comprisesone or more groups selected from the group consisting of an acrylategroup, a cyanoacrylamide group, a cyanoacrylate group, and ahaloacetamide group. In some embodiments, the test compound comprisesone or more chloroacetamide groups (e.g., a substituted or unsubstitutedchloroacetamide). As used here, the term “acrylamide” refers to a groupof Formula I or Formula II:

wherein R¹, R², and R³ are each independently selected from the groupconsisting of H, C₁₋₆ alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, amino, C₁₋₆alkylamino, and di(C₁₋₆ alkyl)amino; and

wherein ring A of Formula II represented a 4-20 memberedheterocycloalkyl or a 5-20 membered heteroaryl group, each of which maybe optionally substituted.

As used herein, the phrase “optionally substituted” means unsubstitutedor substituted. The substituents are independently selected, andsubstitution may be at any chemically accessible position. As usedherein, the term “substituted” means that a hydrogen atom is removed andreplaced by a substituent. A single divalent substituent, e.g., oxo, canreplace two hydrogen atoms. It is to be understood that substitution ata given atom is limited by valency. Suitable substituents include, butare not limited to, C₁₋₆ alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, amino, C₁₋₆alkylamino, and di(C₁₋₆ alkyl)amino.

The term “n-membered” where n is an integer typically describes thenumber of ring-forming atoms in a moiety where the number ofring-forming atoms is n. For example, piperidinyl is an example of a6-membered heterocycloalkyl ring, pyrazolyl is an example of a5-membered heteroaryl ring, pyridyl is an example of a 6-memberedheteroaryl ring, and 1,2,3,4-tetrahydro-naphthalene is an example of a10-membered cycloalkyl group.

Throughout the definitions, the term “Cn-m” indicates a range whichincludes the endpoints, wherein n and m are integers and indicate thenumber of carbons. Examples include C₁₋₄, C₁₋₆, and the like.

As used herein, the term “Cn-m alkyl”, employed alone or in combinationwith other terms, refers to a saturated hydrocarbon group that may bestraight-chain or branched, having n to m carbons. Examples of alkylmoieties include, but are not limited to, chemical groups such asmethyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl,sec-butyl; higher homologs such as 2-methyl-1-butyl, n-pentyl, 3-pentyl,n-hexyl, 1,2,2-trimethylpropyl, and the like. In some embodiments, thealkyl group contains from 1 to 6 carbon atoms, from 1 to 4 carbon atoms,from 1 to 3 carbon atoms, or 1 to 2 carbon atoms.

As used herein, “Cn-m alkenyl” refers to an alkyl group having one ormore carbon-carbon double bonds and having n to m carbons. Examplealkenyl groups include, but are not limited to, ethenyl, allyl,n-propenyl, isopropenyl, n-butenyl, sec-butenyl, and the like. In someembodiments, the alkenyl moiety contains 2 to 6, 2 to 4, or 2 to 3carbon atoms.

As used herein, “Cn-m alkynyl” refers to an alkyl group having one ormore carbon-carbon triple bonds and having n to m carbons. Examplealkynyl groups include, but are not limited to, ethynyl, propyn-1-yl,propyn-2-yl, and the like. In some embodiments, the alkynyl moietycontains 2 to 6, 2 to 4, or 2 to 3 carbon atoms.

As used herein, the term “amino” refers to a group of formula —NH₂.

As used herein, the term “Cn-m alkylamino” refers to a group of formula—NH(alkyl), wherein the alkyl group has n to m carbon atoms. In someembodiments, the alkyl group has 1 to 6, 1 to 4, or 1 to 3 carbon atoms.Examples of alkylamino groups include, but are not limited to,N-methylamino, N-ethylamino, N-propylamino (e.g., N-(n-propyl)amino andN-isopropylamino), N-butylamino (e.g., N-(n-butyl)amino andN-(tert-butyl)amino), and the like.

As used herein, the term “di(Cn-m-alkyl)amino” refers to a group offormula —N(alkyl)₂, wherein the two alkyl groups each has,independently, n to m carbon atoms. In some embodiments, each alkylgroup independently has 1 to 6, 1 to 4, or 1 to 3 carbon atoms.

As used herein, the term “non-selective thiol-reactive compound” or“compound having broad thiol reactivity” refers to a compound comprisingone or more moieties that are non-selectively reactive towards a thiolgroup (e.g., —SH). Exemplary moieties having reactivity towards a thiolgroup include, but are not limited to, acrylamide, dimethylaminoacrylamide, iodoacetamide, chloroacetamide, maleimide, and C—X groups,wherein X is a halogen. In some embodiments, the non-selectivethiol-reactive compound is labeled (e.g., radiolabeled) with heavyisotopes of carbon, oxygen, nitrogen, sulfur, phosphorous, chlorine,bromine, or hydrogen. In some embodiments, the non-selectivethiol-reactive compound or compound having broad thiol reactivitycomprises one or more acrylamide groups, dimethylamino acrylamidegroups, iodoacetamide groups, chloroacetamide groups, maleimide groups,C—X groups, or any combination thereof, wherein X is a halogen.

In some embodiments, the test compound comprises one acrylamide group.In some embodiments, the test compound comprises more than oneacrylamide group. In some embodiments, the test compound comprises oneor more terminal acrylamide groups.

In some embodiments, the test compound comprises one haloacetamidegroup. In some embodiments, the test compound comprises more than onehaloacetamide group. In some embodiments, the test compound comprisesone or more terminal haloacetamide groups.

In some embodiments, the test compound comprises one electrophilicgroup. In some embodiments, the test compound comprises more than oneelectrophilic group. In some embodiments, the test compound comprisesone or more terminal electrophilic groups.

In some embodiments, the test compound is chemically modified tofacilitate affinity-based enrichment of test compound polypeptideconjugates. In some embodiments chemical-modification of the testcompound comprises an affinity tag such as a peptide-epitope, biotin, ordesthiobiotin. In some embodiments the chemical-modification of the testcompound comprises a bio-orthogonal moiety such as an alkyne or azide.

In some embodiments, the polypeptide comprises one or more groupscapable of forming thioether bonds with the test compound (e.g., withone or more sulfur atoms in the test compound). In some embodiments, thepolypeptide comprises one or more groups capable of forming covalentthioether bonds with the test compound. In some embodiments, thepolypeptide comprises one or more acrylamide groups. In someembodiments, the polypeptide comprises one acrylamide group. In someembodiments, the polypeptide comprises more than one acrylamide group.In some embodiments, the polypeptide comprises one or more terminalacrylamide groups.

In some embodiments, the test compound is identified as a kinaseinhibitor or a deubiquitinase inhibitor (e.g., TL11-113). In someembodiments, the test compound is identified as a kinase inhibitor.Example kinase inhibitors include, but are not limited to, JNK-IN-7,HBX-19818, MI-2, TL10-201, THZ531, THZ1, QL-47, ibrutinib, neratinib,afatinib, axitinib, bosutinib, cobimetinib, crizotinib, entrectinib,erlotinib, and the like. In some embodiments, the test compound isselected from the group consisting of JNK-IN-7, HBX-19818, MI-2,TL10-201, THZ531, THZ1, QL-47, ibrutinib, and neratinib. In someembodiments, the test compound is selected from the group consisting ofJNK-IN-7, HBX-19818, MI-2, TL10-201, THZ531, THZ1, QL-47, TL11-113,ibrutinib, and neratinib.

In some embodiments, a reagent having broad thiol reactivity and highyield of thiolated ions, or derivative ions thereof, is used to measurethe binding stoichiometry of the test compound to its target. In someembodiments, the reagent with broad thiol reactivity and high yield ofthiolated ions, or derivative ions thereof, is selected from the groupconsisting of:

-   2-iodo-1-morpholinoethan-1-one;-   1-((2R,6R)-2,6-dimethylmorpholino)-2-iodoethan-1-one;-   1-((2R,6S)-2,6-dimethylmorpholino)-2-iodoethan-1-one;-   1-(2,2-dimethylmorpholino)-2-iodoethan-1-one;-   1-(3,5-dimethylmorpholino)-2-iodoethan-1-one;-   1-(hexahydrocyclopenta[b][1,4]oxazin-4(4aH)-yl)-2-iodoethan-1-one;-   1-(2,3-dihydro-4H-benzo[b][1,4]oxazin-4-yl)-2-iodoethan-1-one;-   N-(4-cyanophenyl)-2-iodoacetamide;-   N-(2,5-dimethylphenyl)-2-iodoacetamide;-   N-(2,5-dimethoxyphenyl)-2-iodoacetamide;-   2-iodo-N-phenylacetamide;-   2-iodo-N-(p-tolyl)acetamide;-   2-iodo-1-(4-methylpiperazin-1-yl)ethan-1-one 2,2,2-trifluoroacetate;-   N-(furan-2-ylmethyl)-2-iodoacetamide;-   2-iodo-N-(1-methyl-1H-imidazol-4-yl)acetamide; and-   N-ethylmaleimide.

In some embodiments, the reagent having broad thiol reactivity isisotopically labeled with heavy isotopes of carbon, oxygen, nitrogen,sulfur, phosphorous, chlorine, bromine, or hydrogen.

In some embodiments, the polypeptide is a protein or a protein fragment.In some embodiments, the polypeptide is a protein. In some embodiments,the polypeptide is a protein fragment.

In some embodiments, the polypeptide is a protein or protein fragmentcomprising from about 4 to about 100 amino acid residues, for example,from about 4 to about 100, about 4 to about 80, about 4 to about 60,about 4 to about 40, about 4 to about 20, about 4 to about 10, about 10to about 100, about 10 to about 80, about 10 to about 60, about 10 toabout 40, about 10 to about 20, about 20 to about 100, about 20 to about80, about 20 to about 60, about 20 to about 40, about 40 to about 100,about 40 to about 80, about 40 to about 60, about 60 to about 100, about60 to about 80, or about 80 to about 100 amino acid residues. In someembodiments, the polypeptide is a protein or protein fragment comprisingfrom about 10 to about 30 amino acid residues. In some embodiments, thepolypeptide is a protein comprising from about 10 to about 30 amino acidresidues. In some embodiments, the polypeptide is a protein fragmentcomprising from about 10 to about 30 amino acid residues. In someembodiments, the polypeptide is a kinase or a kinase fragment. In someembodiments, the polypeptide is a kinase. In some embodiments, thepolypeptide is a kinase fragment.

In some embodiments, the polypeptide is a kinase, a kinase fragment, adeubiquitinase, or a deubiquitinase fragment. In some embodiments, thepolypeptide is a kinase or deubiquitinase selected from the groupconsisting of JNK2, JAK3, CDK7, CDK12, TAK1, ITK, USP-7, and EGFR, or afragment thereof.

In some embodiments, the polypeptide is a kinase selected from the groupconsisting of JNK2, JAK3, CDK7, CDK12, ITK, USP-7, and EGFR, or afragment thereof. In some embodiments, the polypeptide is a kinase or akinase fragment comprising from about 10 to about 100 amino acidresidues, for example, from about 10 to about 100, about 10 to about 80,about 10 to about 60, about 10 to about 40, about 10 to about 20, about20 to about 100, about 20 to about 80, about 20 to about 60, about 20 toabout 40, about 40 to about 100, about 40 to about 80, about 40 to about60, about 60 to about 100, about 60 to about 80, or about 80 to about100 amino acid residues. In some embodiments, the polypeptide is akinase or a kinase fragment comprising from about 10 to about 30 aminoacid residues.

In some embodiments, the polypeptide comprises an amino acid sequencehaving at least 90% sequence identity to a sequence selected from thegroup consisting of:

(SEQ ID NO: 1) L-M-D-A-N-L-C-Q-V-I-Q-M-E; (SEQ ID NO: 2)L-V-M-E-Y-L-P-S-G-C-L-R; (SEQ ID NO: 3)M-A-P-P-D-L-P-H-W-Q-D-C-H-E-L-W-S-K; (SEQ ID NO: 4)H-G-C-L-S-D-Y-L-R-S-Q-R-G-L-F-A-A-E; (SEQ ID NO: 5)Y-F-S-N-R-P-G-P-T-P-G-C-Q-L-P-R-P-N-C-P-V-E-T-L-K; (SEQ ID NO: 6)G-C-L-L-D-Y-V-R; (SEQ ID NO: 7) F-G-L-C-S-G-P-A-D-T-G-R; (SEQ ID NO: 8)Y-M-A-N-G-C-L-sL-N-Y-L-R; (SEQ ID NO: 9)I-C-D-F-G-T-A-C-D-I-Q-T-H-M-T-N-N-K; and (SEQ ID NO: 10)Y-F-S-N-R-P-G-P-T-P-G-C-Q-L-P-(13C6-15N4)R-P-N-C- P-V-E-T-L-K.

In some embodiments, the polypeptide comprises an amino acid sequencehaving at least 90% sequence identity to a sequence selected from thegroup consisting of:

(SEQ ID NO: 1) L-M-D-A-N-L-C-Q-V-I-Q-M-E; (SEQ ID NO: 2)L-V-M-E-Y-L-P-S-G-C-L-R; (SEQ ID NO: 3)M-A-P-P-D-L-P-H-W-Q-D-C-H-E-L-W-S-K; (SEQ ID NO: 4)H-G-C-L-S-D-Y-L-R-S-Q-R-G-L-F-A-A-E; (SEQ ID NO: 5)Y-F-S-N-R-P-G-P-T-P-G-C-Q-L-P-R-P-N-C-P-V-E-T-L-K; (SEQ ID NO: 6)G-C-L-L-D-Y-V-R; (SEQ ID NO: 7) F-G-L-C-S-G-P-A-D-T-G-R; and(SEQ ID NO: 8) Y-M-A-N-G-C-L-sL-N-Y-L-R.

In some embodiments, the polypeptide comprises an amino acid sequencehaving at least 60% (e.g., at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 99%, or 100%) sequenceidentity to a sequence selected from the group consisting of SEQ IDNOs:1-8.

The present application further provides an analytical method (e.g., ahigh throughput method), comprising:

-   -   i) contacting a first mixture comprising one or more test        compounds with a second mixture comprising one or more        polypeptides to form a third mixture comprising one or more        compound-polypeptide conjugates, wherein each of the        compound-polypeptide conjugates comprise one or more thioether        bonds;    -   ii) analyzing the third mixture using a mass spectrometry assay;    -   iii) detecting one or more thiolated or other compound-specific        fragment ions (e.g., derivative ions as described herein)        produced in the mass spectrometry assay; and    -   iv) identifying that one or more of the test compounds binds        irreversibly to one or more of the polypeptides based on the        detection of a thiolated or other inhibitor-specific fragment        ion (e.g., derivative ions as described herein) in the mass        spectrometry assay.    -   v) quantifying compound selectivity for polypeptide by varying        the concentration of compound and using stable isotope labels to        quantify extent of compound-polypeptide formation at each        concentration.

In some embodiments, the analytical method (e.g., a high throughputmethod), comprises:

-   -   i) contacting a first mixture comprising one or more test        compounds with a second mixture comprising one or more        polypeptides to form a third mixture comprising one or more        compound-polypeptide conjugates, wherein each of the        compound-polypeptide conjugates comprise one or more thioether        bonds;    -   ii) analyzing the third mixture using a mass spectrometry assay;    -   iii) detecting one or more thiolated ions produced in the mass        spectrometry assay; and    -   iv) identifying that one or more of the test compounds binds        irreversibly to one or more of the polypeptides based on the        detection of the one or more thiolated ions in the mass        spectrometry assay.

In some embodiments, the analytical method (e.g., a high throughputmethod), comprises:

-   -   i) contacting a first mixture comprising one or more test        compounds with a second mixture comprising one or more        polypeptides to form a third mixture comprising one or more        compound-polypeptide conjugates, wherein each of the        compound-polypeptide conjugates comprise one or more thioether        bonds;    -   ii) contacting the third mixture with a fourth mixture        comprising one or more chemically-modified analogs of the one or        more test compounds contained in the first mixture to form a        fifth mixture comprising one or more test compound-polypeptide        conjugates and one or more chemically-modified test        compound-polypeptide conjugates; wherein each of the test        compound-polypeptide conjugates and chemically-modified test        compound polypeptide conjugates comprise one or more thioether        bonds;    -   iii) preparing a sixth mixture from the fifth mixture by        application of standard click chemistry or other biorthogonal        chemistry schemes to attach an affinity handle, followed by        biochemical purification methods to enrich the        chemically-modified, tagged test compound-polypeptide        conjugates;    -   iv) analyzing the sixth mixture using a mass spectrometry assay;    -   v) detecting one or more thiolated ions produced in the mass        spectrometry assay; and    -   vi) identifying that one or more of the chemically-modified test        compounds binds irreversibly to one or more of the polypeptides        based on the detection of the one or more thiolated ions in the        mass spectrometry assay.

The present application further provides an analytical method (e.g., ahigh throughput method), comprising:

-   -   i) contacting a first mixture comprising one or more test        compounds with a second mixture comprising one or more        polypeptides to form a third mixture comprising one or more        compound-polypeptide conjugates, wherein each of the        compound-polypeptide conjugates comprise one or more thioether        bonds;    -   ii) contacting the third mixture with a fourth mixture        comprising one or more chemically-modified analogs of the one or        more test compounds contained in the first mixture to form a        fifth mixture comprising one or more test compound-polypeptide        conjugates and one or more chemically-modified test        compound-polypeptide conjugates; wherein each of the test        compound-polypeptide conjugates and chemically-modified test        compound polypeptide conjugates comprise one or more thioether        bonds;    -   iii) preparing a sixth mixture from the fifth mixture, wherein        the sixth mixture comprises one or more chemically-modified test        compound-polypeptide conjugates comprising one or more affinity        tags;    -   iv) analyzing the sixth mixture using a mass spectrometry assay;    -   v) detecting one or more thiolated ions produced in the mass        spectrometry assay; and    -   vi) identifying that one or more of the chemically-modified test        compounds binds irreversibly to one or more of the polypeptides        based on the detection of the one or more thiolated ions in the        mass spectrometry assay.

In some embodiments, the one or more chemically-modified analogs eachcomprise an alkyne or azide moiety.

In some embodiments, preparation of the sixth mixture comprises reactingthe one or more chemically-modified analogs of the fifth mixture underconditions of suitable for performing click chemistry or biorthogonalchemistry to attach an affinity handle to the one or morechemically-modified analogs, thereby producing the chemically-modifiedtest compound-polypeptide conjugates comprising one or more affinitytags.

In some embodiments, the analytical method further comprisingbiochemically purifying the sixth mixture to enrich the sixth mixture inthe chemically-modified, tagged test compound-polypeptide conjugates,prior to the analyzing of step iv).

In some embodiments, the method further comprises quantifying compoundselectivity for polypeptide by varying the concentration of compound andusing stable isotope labels to quantify extent of compound-polypeptideformation at each concentration.

In some embodiments, the method further comprises the use ofchemically-modified analogs of each of the one or more test compounds ina competition-format, wherein the native (i.e., not chemically-modified)test compounds are first added (e.g., at varying concentration), to amixture of one or more polypeptides. This first addition step isfollowed by addition of a fixed-concentration of chemically-modifiedanalogs of each of the one or more test compounds. Standard chemical(e.g., click chemistry) and biochemical methods are used to connectaffinity handles and purify chemically-modified, affinity-tagged testcompound-polypeptide conjugates. In some embodiments, test compoundselectivity is established by use of stable isotope labels to quantifythe extent of compound-polypeptide formation at each concentration ofnative (i.e., not chemically-modified) test compound used.

The present application further provides an analytical method,comprising:

-   -   i) contacting a first mixture comprising one or more test        compounds with a second mixture comprising one or more        polypeptides to form a third mixture comprising one or more        compound-polypeptide conjugates, wherein each of the        compound-polypeptide conjugates comprise one or more thioether        bonds;    -   ii) analyzing the third mixture using a mass spectrometry assay;    -   iii) detecting one or more thiolated ions produced in the mass        spectrometry assay; and

iv) identifying that one or more of the test compounds bindsirreversibly to one or more of the polypeptides based on the detectionof the one or more thiolated ions in the mass spectrometry assay.

In some embodiments, the method further comprises digesting the testcompound-polypeptide conjugate prior to performing the mass spectrometryassay of step ii).

In some embodiments, the digesting comprises reacting the testcompound-polypeptide conjugate with trypsin or another proteolyticenzyme in the presence of a third solvent component. In someembodiments, the digesting comprises reacting the testcompound-polypeptide conjugate with trypsin in the presence of a thirdsolvent component.

In some embodiments, the third solvent component comprises a pH 7-9buffer agent (e.g., triethylammonium bicarbonate, PBS, HEPES, and thelike). In some embodiments, the third solvent component comprisesammonium bicarbonate. In some embodiments, the third solvent componentcomprises 100 mM ammonium bicarbonate in water. In some embodiments, thethird solvent component further comprises a chaotropic agent (e.g., ureaor GuHCl). In some embodiments, the third solvent component furthercomprises Rapigest or other mass spectrometry-compatible surfactant.

In some embodiments, the test compounds in the first mixture can be thesame and/or have the same characteristics of the test compoundsdescribed above (e.g., each of the test compounds comprises one or morethiol-reactive groups including, but not limited to, acrylamide groups,dimethylamino acrylamide groups, iodoacetamide groups, chloroacetamidegroups, maleimide groups, or reactive C—X groups, wherein X is ahalogen). In some embodiments, each of the test compounds are encodedwith heavy isotopes of carbon, oxygen, nitrogen, sulfur, phosphorous,chlorine, bromine, or hydrogen. In some embodiments, the polypeptides inthe second mixture can be the same and/or have the same characteristicsof the polypeptides described above.

In some embodiments, the method further comprises reacting one or moreacrylamide compounds with beta-mercaptoethanol or similar Michael donorto form one or more inactivated test compound, prior to the contactingof step i).

In some embodiments, the method further comprises reacting one or moretest compounds, each containing a thiol-reactive moiety, with a secondcompound containing a nucleophilic thiol group to form one or more testcompound thioether conjugates, prior to the contacting of step i). Insome embodiments, the second compound is selected from the groupconsisting of beta-mercaptoethanol, glutathione, 2-mercaptobenzoic acid,and hydrogen sulfide.

In some embodiments, the method further comprises reacting one or moreacrylamide compounds with a thiolated compound to form one or moreacrylamide thiolated derivatives, prior to the contacting of step i).

In some embodiments, the method further comprises analyzing the one ormore test compound thioether conjugates in a mass spectrometry assayprior to the contacting step i). In some embodiments, analyzing the testcompound thioether conjugates comprises generating a database offragment ion spectra comprising the mass spectra of each of the one ormore test compound thioether conjugates. In some embodiments, the methodfurther comprises gas-phase isolation and fragmentation (e.g., MS/MS/MSor MS3) of the one or more of the thiolated ions, or derivative ionsthereof, formed during MS/MS of the one or more test compound thioetherconjugates.

In some embodiments, the method further comprises analyzing the one ormore inactivated test compounds in a mass spectrometry assay.

In some embodiments, the method further comprises analyzing the one ormore acrylamide thiolated derivatives in a mass spectrometry assay.

In some embodiments, analyzing the one or more inactivated testcompounds comprises generating a database of mass spectral fragment ionsby performing MS/MS/MS on each inactivated test compound.

In some embodiments, analyzing the one or more acrylamide thiolatedderivatives comprises generating a database of fragment ion spectracomprising the mass spectra of each of the one or more acrylamidethiolated derivatives.

In some embodiments, the method further comprises gas-phase isolationand fragmentation (e.g., MS/MS/MS or MS3) of the one or more thiolatedions, or derivative ions thereof, detected in step iii).

In some embodiments, the method further comprises performing MS/MS/MS onthe one or more thiolated ions detected as a result of MS/MS in stepiii).

In some embodiments, the method further comprises isolating the one ormore thiolated ions after the detecting of step iii).

In some embodiments, the method further comprises performing a massspectrometry assay on the one or more isolated thiolated ions prior tothe identifying of step iv).

In some embodiments, the identifying of step iv) further comprisesidentifying a mass spectrum in the database of fragment ion spectra(e.g., MS/MS/MS or MS3 fragment ion spectra) from the one or more testcompound thioether conjugates that is substantially identical to thefragment ion spectrum derived from the gas-phase isolation andfragmentation analysis (e.g., MS/MS/MS or MS3) performed on thethiolated ion detected in step iii).

In some embodiments, the identifying of step iv) further comprisesidentifying a mass spectrum in the database of fragment ions derivedfrom the inactivated test compounds that is substantially identical tothe mass spectrum of the isolated thiolated ion.

In some embodiments, the identifying of step iv) further comprisesidentifying a mass spectrum in the database of fragment ion spectra thatis substantially identical to the mass spectrum of the isolatedthiolated ion. In some embodiments, the thiolated compound isβ-mercaptoethanol.

In some embodiments, the first mixture comprises more than one testcompound. In some embodiments, the second mixture comprises more thanone polypeptide. In some embodiments, the first mixture comprises morethan one test compound and the second mixture comprises more than onepolypeptide.

In some embodiments, the first mixture comprises from about 2 to about100,000 different test compounds, for example, from about 2 to about100,000, about 2 to about 75,000, about 2 to about 50,000, about 2 toabout 10,000, about 2 to about 5000, about 2 to about 1000, about 2 toabout 100, about 2 to about 50, about 50 to about 100,000, about 50 toabout 75,000, about 50 to about 50,000, about 50 to about 10,000, about50 to about 5000, about 50 to about 1000, about 50 to about 100, about100 to about 100,000, about 100 to about 75,000, about 100 to about50,000, about 100 to about 10,000, about 100 to about 5000, about 100 toabout 1000, about 1000 to about 100,000, about 1000 to about 75,000,about 1000 to about 50,000, about 1000 to about 10,000, about 1000 toabout 5000, about 5000 to about 100,000, about 5000 to about 75,000,about 5000 to about 50,000, about 5000 to about 10,000, about 10,000 toabout 100,000, about 10,000 to about 75,000, about 10,000 to about50,000, about 50,000 to about 100,000, about 50,000 to about 75,000, orabout 75,000 to about 100,000 test compounds.

In some embodiments, the second mixture is a biological sample, forexample, a cell, a tissue, a bone, a cell sample, a tissue sample, abone sample, and the like. In some embodiments, the second mixture is acell sample.

In some embodiments, the first mixture and the second mixture are usefulfor performing a high throughput assay. The high throughput assay can beused to identify effective covalent probes for certain proteins (e.g.,cysteine-containing proteins) in a cell sample.

The present application further provides an analytical method (e.g., ahigh throughput method) comprising:

-   -   i) reacting one or more acrylamide compounds with a        thiol-containing compound to form one or more inactivated test        compounds;    -   ii) analyzing the one or more inactivated test compounds in a        mass spectrometry assay;    -   iii) generating a database of fragment ion spectra comprising        the mass spectra of each of the one or more inactivated test        compounds;    -   iv) contacting a first mixture comprising more than one test        compound, wherein each test compound may be introduced at        different concentrations, with a second mixture comprising more        than one polypeptide to form a third mixture comprising more        than one compound-polypeptide conjugate, wherein each of the        compound-polypeptide conjugates comprise one or more thioether        bonds;    -   v) analyzing the third mixture using a mass spectrometry assay;    -   vi) detecting one or more thiolated or other compound-specific        fragment ions produced in the mass spectrometry assay;    -   vii) isolating the one or more thiolated ions in the mass        spectrometer;    -   viii) performing a mass spectrometry assay on the one or more        isolated thiolated ions;    -   ix) comparing the mass spectra of the one or more thiolated ions        to the database of fragment ion spectra; and    -   x) identifying that one or more of the test compounds binds        irreversibly to one or more of the polypeptides based on the        detection of one or more thiolated ions in the mass spectrometry        assay of step v) and determining that the mass spectrum in the        database of fragment ion spectra is substantially identical to        the mass spectrum of the isolated thiolated ion of step viii).

In some embodiments, the analytical method comprises:

-   -   i) reacting one or more acrylamide compounds with a        thiol-containing compound to form one or more acrylamide        thiolated derivatives;    -   ii) analyzing the one or more acrylamide thiolated derivatives        in a mass spectrometry assay;    -   iii) generating a database of fragment ion spectra comprising        the mass spectra of each of the one or more acrylamide thiolated        derivatives;    -   iv) contacting a first mixture comprising more than one test        compound with a second mixture comprising more than one        polypeptide to form a third mixture comprising more than one        compound-polypeptide conjugate, wherein each of the        compound-polypeptide conjugates comprise one or more thioether        bonds;    -   v) analyzing the third mixture using a mass spectrometry assay;    -   vi) detecting one or more thiolated ions produced in the mass        spectrometry assay;    -   vii) isolating the one or more thiolated ions;    -   viii) performing a mass spectrometry assay on the one or more        isolated thiolated ions;    -   ix) comparing the mass spectra of the one or more thiolated ions        to the database of fragment ion spectra; and    -   x) identifying that one or more of the test compounds binds        irreversibly to one or more of the polypeptides based on the        detection of one or more thiolated ions in the mass spectrometry        assay of step v) and the identification a mass spectrum in the        database of fragment ion spectra that is substantially identical        to the mass spectrum of the isolated thiolated ion of step        viii).

The present application further provides an analytical method,comprising:

-   -   i) reacting one or more test compounds each containing a        thiol-reactive moiety with a second compound containing a thiol        group to form one or more test compound thioether conjugates;    -   ii) analyzing the one or more test compound thioether conjugates        in a mass spectrometry assay;    -   iii) generating a database of fragment ion spectra comprising        the mass spectra derived from the mass spectrometry assay (e.g.,        MS/MS/MS or MS3) performed on each of the one or more test        compound thioether conjugates;    -   iv) contacting a first mixture comprising more than one test        compound with a second mixture comprising more than one        polypeptide to form a third mixture comprising more than one        test compound-polypeptide conjugate, wherein each of the test        compound-polypeptide conjugates comprise one or more thioether        bonds;    -   v) analyzing the third mixture using a mass spectrometry assay;    -   vi) detecting one or more thiolated ions, or derivative ions        thereof, produced in the mass spectrometry assay;    -   vii) performing gas phase isolation and MS/MS/MS or MS3 analysis        on the one or more thiolated ions, or derivative ions thereof,        to generate fragment ion spectra;    -   viii) comparing the fragment ion spectra generated in step vii)        with the database of fragment ion spectra generated in step        iii); and    -   ix) identifying that one or more of the test compounds binds        irreversibly to one or more of the polypeptides based on the        detection of one or more thiolated ions, or derivative ions        thereof, in the mass spectrometry assay of step v) and the        identification of a mass spectrum in the database of fragment        ion spectra that is substantially identical to the mass spectrum        of the thiolated ion, or derivative ion thereof, generated in        step iii).

In some embodiments, the fragment ion spectra generated in step vii) arediagnostic of the one or more test compounds.

The gas phase fragmentation behavior specific to each probe can be usedto develop targeted mass spectrometry assays. These assays may be usedwhen the target and compound are known, for example, to validate thestoichiometry of target engagement.

Accordingly, the present application further provides an analyticalmethod, comprising:

-   -   i) contacting a test compound with a second mixture comprising        one or more polypeptides to form a third mixture comprising one        or more compound-polypeptide conjugates, wherein each of the        compound-polypeptide conjugates comprise one or more thioether        bonds;    -   ii) treating separate aliquot of the second mixture with a        vehicle control such as DMSO;    -   iii) treating both mixtures with a compound having        non-selective, broad thiol reactivity, which forms a thioether        linkage; examples of such compounds include acrylamide or        maleimide or N-functionalized maleimide; in one embodiment the        non-selective thiol reactive compounds are encoded with stable        isotope labels such as ¹⁵N, ¹³C, or ¹⁸O;    -   iv) combining both mixtures; in one embodiment the target of        interest is further enriched from the combined mixture using        immunoaffinity capture reagents;    -   v) digesting the combined mixture or the enriched mixture        resulting from immunoaffinity capture with trypsin or other        proteolytic enzyme;    -   vi) analyzing the digested peptides using a targeted mass        spectrometry assay;    -   vii) detecting one or more thiolated or other specific fragment        ions produced by the thioether linkage between the target and        the non-selective thiol-reactive compound in the mass        spectrometry assay; and    -   viii) determining target engagement stoichiometry for the        compound-polypeptide conjugate based on the ratio of thiolated        or other specific fragment ions resulting from the        non-selective, thiol-reactive compound detected in mixtures        originally reacted with test compound or DMSO (e.g., vehicle        control).

In some embodiments, the analytical method comprises:

-   -   i) contacting a test compound with a first mixture comprising        one or more polypeptides to form a second mixture comprising one        or more compound-polypeptide conjugates, wherein each of the        compound-polypeptide conjugates comprise one or more thioether        bonds;    -   ii) treating an aliquot of the first mixture with a vehicle        control to form a third mixture;    -   iii) treating both the second and third mixtures with        isotopically labeled or unlabeled broad thiol-reactive compounds        to form fourth and fifth mixtures comprising one or more        thioether bonds between the broad thiol reactive compounds and        the one or more polypeptides;    -   iv) combining the fourth and fifth mixtures to form a combined        mixture;    -   v) digesting the combined polypeptide mixture (e.g., in the        presence of trypsin or a digestive enzyme such as a proteolytic        enzyme) to form a mixture of (a) one or more test        compound-peptide conjugates, (b) one or more broad        thiol-reactive compound-peptide conjugates, and (c) one or more        isotopically labeled broad thiol-reactive compound-peptide        conjugates, whereby each conjugate is formed through one or more        thioether bonds;    -   vi) analyzing the peptides using a mass spectrometry assay        (e.g., a targeted mass spectrometry assay);    -   vii) detecting one or more thiolated ions, or derivative ions        thereof, produced in the mass spectrometry assay; and    -   viii) determining target engagement stoichiometry for the test        compound-polypeptide conjugate based on the ratio of thiolated        ions, or derivative ions thereof, derived from the isotopically        labeled and unlabeled broad thiol reactive compound-peptide        conjugates, produced in the targeted mass spectrometry assay.

In some embodiments, the method further comprises:

-   -   iv-a) enriching the compound-polypeptide conjugate in the        combined mixture using one or more immunoaffinity capture        reagents, wherein step iv-a) is performed after step iv) and        prior to step v).

The present application further provides an analytical method,comprising:

-   -   i) contacting a test compound with a first mixture comprising        one or more polypeptides to form a second mixture comprising one        or more test compound-polypeptide conjugates, wherein each of        the test compound-polypeptide conjugates comprise one or more        thioether bonds;    -   ii) treating an aliquot of the first mixture with a vehicle        control to form a third mixture;    -   iii) treating the third mixture with a broad thiol-reactive        compound to form a fourth mixture comprising one or more broad        thiol-reactive compound-polypeptide conjugates formed through        one or more thioether bonds;    -   iv) treating the second mixture with a broad thiol-reactive        compound labeled with one or more stable isotopes selected from        the group consisting of ¹⁵N, ¹³C, and ¹⁸O to form a fifth        mixture comprising one or more test compound-polypeptide        conjugates and one or more isotopically labeled broad        thiol-reactive compound-polypeptide conjugates, whereby each        conjugate is formed through one or more thioether bonds;    -   v) combining the fourth and fifth mixtures to form a combined        mixture;    -   vi) enzymatically digest the combined mixture of polypeptides        and polypeptide-conjugates to form a mixture of peptides        comprising a combination of (i) one or more test        compound-peptide conjugates, (ii) one or more broad        thiol-reactive compound-peptide conjugates, and (iii) one or        more isotopically labeled broad thiol-reactive compound-peptide        conjugates, whereby each conjugate is formed through one or more        thioether bonds;    -   vi) analyzing the combined mixture of peptides using a targeted        mass spectrometry assay;    -   vii) detecting one or more thiolated ions, or derivative ions        thereof, produced in the mass spectrometry assay; and    -   viii) determining target engagement stoichiometry for the test        compound-polypeptide conjugate based on the ratio of thiolated        ions, or derivative ions thereof, derived from the isotopically        labeled and unlabeled broad thiol reactive compound-peptide        conjugates, produced in the targeted mass spectrometry assay.

In some embodiments, the method further comprises:

-   -   iv-a) enriching the compound-polypeptide conjugate in the        combined mixture using one or more immunoaffinity capture        reagents, wherein step iv-a) is performed after step iv) and        prior to step v).

In some embodiments, the vehicle control is DMSO.

In some embodiments, the compound having non-selective, broad thiolreactivity comprises an acrylamide group, a maleimide group, aN-functionalized maleimide, or any combination thereof. In someembodiments, the compound having non-selective, broad thiol reactivitycomprises one or more acrylamide groups, dimethylamino acrylamidegroups, iodoacetamide groups, chloroacetamide groups, maleimide groups,or reactive C—X groups, wherein X is a halogen. In some embodiments,each of the test compounds are encoded with heavy isotopes of carbon,oxygen, nitrogen, sulfur, phosphorous, chlorine, bromine, or hydrogen.

In some embodiments, the compound having non-selective, broad thiolreactivity is encoded with stable isotope labels selected from the groupconsisting of ¹⁵N, ¹³C, and ¹⁸O.

The present application further provides a compound or ion describedherein. In some embodiments, the compound or ion is prepared accordingto one or more of the methods described herein. In some embodiments, thecompound or ion is selected from the group of compounds provided inTable A.

TABLE A

In some embodiments, a compound or ion provided in Table A is anisotopically labeled compound or ion.

As used herein, a compound or ion described herein as “isotopicallylabeled” (e.g., a compound labeled with one or more heavy isotopes; acompound comprising one or more heavy isotopes; and the like) is acompound or ion wherein one or more atoms are replaced or substituted byan atom having an atomic mass or mass number different from the mostabundant atomic mass or mass number typically found in nature (i.e.,naturally occurring). Exemplary isotopes that may be incorporated incompounds or ions of the present invention include but are not limitedto ²H (also written as D for deuterium), ³H (also written as T fortritium), ¹³C, ¹⁵N, ¹⁸O, ³⁵S, ³⁶Cl, ⁸²Br, ⁷⁵Br, ⁷⁶Br, ⁷⁷Br.

In some embodiments, a compound provided in Table A comprises one ormore heavy isotopes of carbon, oxygen, nitrogen, sulfur, phosphorous,chlorine, bromine, or hydrogen.

In some embodiments, one or more nitrogen atoms of a compound providedin Table A are replaced by ¹⁵N.

In some embodiments, one or more carbon atoms of a compound provided inTable A are replaced by ¹³C.

In some embodiments, one or more oxygen atoms of a compound provided inTable A are replaced by ¹⁸O.

As described herein, the reactions for preparing the test compounds,polypeptides, and test compound-polypeptide conjugates described hereincan be carried out in suitable solvents which can be readily selected byone of skill in the art of organic synthesis. Suitable solvents can besubstantially non-reactive with the starting materials (reactants), theintermediates, or products at the temperatures at which the reactionsare carried out, (e.g., temperatures which can range from the solvent'sfreezing temperature to the solvent's boiling temperature). A givenreaction can be carried out in one solvent or a mixture of more than onesolvent. Depending on the particular reaction step, suitable solventsfor a particular reaction step can be selected by the skilled artisan.

The expressions, “ambient temperature” and “room temperature” or “rt” asused herein, are understood in the art, and refer generally to atemperature, e.g. a reaction temperature, that is about the temperatureof the room in which the reaction is carried out, for example, atemperature from about 20° C. to about 30° C. (such as about 25° C.).

Preparation of test compounds described herein can involve theprotection and deprotection of various chemical groups. The need forprotection and deprotection, and the selection of appropriate protectinggroups, can be readily determined by one skilled in the art. Thechemistry of protecting groups can be found, for example, in T. W.Greene and P. G. M. Wuts, Protective Groups in Organic Synthesis, 3^(rd)Ed., Wiley & Sons, Inc., New York (1999).

Reactions can be monitored according to any suitable method known in theart. For example, product formation can be monitored by spectroscopicmeans, such as nuclear magnetic resonance spectroscopy (e.g., ¹H or¹³C), infrared spectroscopy, spectrophotometry (e.g., UV-visible), massspectrometry, or by chromatographic methods such as high performanceliquid chromatography (HPLC), liquid chromatography-mass spectroscopy(LCMS), or thin layer chromatography (TLC). The test compounds,polypeptides, and test compound-polypeptide conjugates described hereincan be purified by those skilled in the art by a variety of methods,including for example, high performance liquid chromatography (HPLC) andnormal phase silica chromatography.

EXAMPLES

The invention will be described in greater detail by way of specificexamples. The following examples are offered for illustrative purposes,and are not intended to limit the invention in any manner. Those ofskill in the art will readily recognize a variety of non-criticalparameters which can be changed or modified to yield essentially thesame results.

Example 1. Preparation of Model Mixtures

Model peptides were synthesized using standard Fmoc chemistry known inthe art and purified by reversed phase HPLC. Test compounds (i.e.,peptide inhibitors) were synthesized as previously described (see e.g.,Kwiatkowski et al, Nature, 2014, 511:616-620; Tan et al, J. Med. Chem.2015, 58:6589-6606; and Zhang et al, Chem. Biol. 2012, 19:140-154) orobtained from commercial sources. All other compounds, unless notedotherwise, were obtained from Sigma-Aldrich.

K562 tryptic peptide aliquots were prepared as described (see e.g.,Ficarro et al, Mol. Cell Proteomics, 2011, 10, 0111, 011064). Peptidesmodified with inhibitors were produced by incubating a 10-fold molarexcess of inhibitor with synthetic peptides (F-G-L-C-S-G-P-A-D-T-G-R(SEQ ID NO: 7) or Y-M-A-N-G-C-L-sL-N-Y-L-R (SEQ ID NO: 8), sL=¹⁵N-1,¹³C-6 leucine) or reduced, desalted tryptic bovine serum albuminpeptides in 1:1 DMSO/100 mM triethylammonium bicarbonate, pH 8.5 at 37°C. overnight. Some combinations required incubation at 60° C. overnightto produce useful levels of derivatization.

Example 2. Collision Energy Profiling

Synthetic covalently modified peptides were diluted 1:200 with 50%MeCN/water with 1% acetic acid and directly infused into a QExactive HFmass spectrometer at a flow rate of 3 μL/min using the standard ionsource (spray voltage=4 kV, sheath gas=1). Spectra at a range ofcollision energies (e.g., 10-100 eV) were manually acquired in tune modefrom m/z 100 to 1500 at a resolution of 15000. Intensities of ions wereextracted and exported using multiplierz scripts. Normalized intensityvalues were derived and plotted using R (version 3.0.2).

Example 3. Protein Labeling and Nanoflow LC-MS/MS

Recombinant JNK, JAK3, CDK12, CDK7, EGFR, USP-7 and ITK proteins werelabeled, digested, and analyzed as previously described (see e.g.,Kwiatkowski et al, Nature, 2014, 511:616-620; Tan et al, J. Med. Chem.2015, 58:6589-6606; and Zhang et al, Chem. Biol. 2012, 19:140-154).

Example 4. Precursor Ion Scanning

Model peptides F-G-L-C-S-G-P-A-D-T-G-R (SEQ ID NO: 7; 250 fmol) andY-M-A-N-G-C-L-sL-N-Y-L-R (SEQ ID NO: 8; 500 fmol) conjugated to QL47 oribrutinib were spiked into 500 ng K562 tryptic peptides and analyzed ona QTRAP 5500 mass spectrometer (AB Sciex, Framingham, Mass.). To detectQL47 modified peptides, the mass spectrometer conducted scan cycles ofQ3MS followed by precursors of 482 (CE=30) or 394 (CE=40). For ibrutinibmodified peptides, the mass spectrometer scanned for precursors of 474(CE=32) or 304 (CE=50).

Example 5. Mass Spectrometry Analysis

Previously characterized acrylamide-warhead kinase probes (i.e., testcompounds) were selected for analysis, and are shown below in Table 1.

TABLE 1 Test Primary Com- Target Reactive pounds Proteins GroupReference JNK-IN-7 JNK2/3 Acrylamide* Zhang et al, Chem. Biol. 2012,19:140-154 THZ1 CDK7 Acrylamide* Kwiatkowski et al, Nature, 2014,511:616-620 THZ531 CDK12/13 Acrylamide* Kwiatkowski et al. TL10-201 JAK3Acrylamide Tan et al, J. Med. Chem. 2015, 58:6589-6606 Ibrutinib BTK/ITKAcrylamide Pan et al, ChemMedChem, 2007, 2:58-61 Neratinib HER2/EGFRAcrylamide* Burstein et al, J. Clin. Oncol. 2010, 28:1301- 1307 QL-47BTK Acrylamide Wu et al, ACS Chem. Biol. 2014, 9:1086-1091 HBX- USP7C-Cl Reverdy et al, Chem. Biol. 19818 2012, 19:467-477 MI-2 MALT-1Chloroacetamide Fontan et al, Cancer Cell, 2012, 22, 812-824 *denotes adimethylamino group functionalized near the warhead.

Each test compound was incubated separately with the target protein,after which mass spectrometry was used to detect intact testcompound-protein conjugates, and to verify the number of covalentmodifications per protein. Next, each protein was digested, theresulting peptides were desalted, and nanoflow LC-MS/MS data wasacquired. FIG. 1A shows a JNK-derived peptide (L-M-D-A-N-L-C-Q-V-I-Q-M-E(SEQ ID NO.: 1)) containing Cys116 modified by JNK-IN-7. It was notedthat several ions detected in the MS/MS spectrum could not be assignedto canonical b- or y-type fragments which result from gas-phase cleavageof peptide amide bonds. Further investigation showed that these andother fragments were derived from the test compound, as shown in Table2.

TABLE 2 Test Compounds and Inhibitor-Specific Fragment Ion Identified inMS/MS Spectrum of JNK-IN-7 Modified JNK2 Target Peptide ION PROPOSEDSTRUCTURE FORMULA Ion m/z Test Compound (JNK-IN-7)

C28 H27 N7 O2 N/A TH (Thiolated ion)

C28 H29 N7 O2 S1 528.2176 (H+) RMA (Retro- Michael addition)

C28 H27 N7 O2 494.2299 (H+) RMA-DMAE-1 (Retro- Michael addition/dimethylamine elimation type 1)

C26 H22 N6 O2 451.1877 (H +) RMA-DMAE-2 (Retro- Michael addition/dimethylamine elimination type 2)

C26 H20 N6 O2 449.1721 (H+) TH-DMAE (Thiolated ion with dimethylamineelimination)

C26 H22 N6 O2 S1 483.1598 (H+) iy1

C22 H18 N6 O1 383.1615 (H+) iy2

C15 H13 N5 264.1244 (H+) ib2 RMA-DMAE2 1b2 with retro- Michaeladdition-and type 2 dimethylamine elimination

C11 H8 N1 O2 186.0555 ib1

C6 H10 N1 O1 112.0762

In particular, one fragment (FIG. 1A, “*” and Table 2, row labeled “TH(Thiolated)”) corresponded to cleavage of the peptide-probe conjugate,yielding an ion containing the intact inhibitor in addition to thetarget cysteine thiol. These data further confirm Cys116 on JNK as thesite of covalent modification. Other fragments resulted from variouselimination reactions, forming “derivative ions” as described throughoutthe present application, for example, of the dimethylamino group or theinhibitor itself (retro-Michael addition), or cleavage of amide bondswithin the inhibitor. The nomenclature provided in Table 2 describeseach amide linkage in an inhibitor which is numbered based on proximityto the cysteine thiol (e.g., iy₁ and ib₁, iy₂ and ib₂, etc.). Inaddition, neutral loss of the inhibitor was observed from canonical b-and y-type ions via retro-Michael addition, as shown in Table 2.

The same fragmentation pathways (i.e., formation of thiolated ions, orderivative ions thereof) were then characterized in twowell-characterized acrylamide probes, ibrutinib and neratinib, clinicaldrugs which target BTK and HER2/EGFR, respectively, as shown in FIGS.1B-1C and FIGS. 8A-8E. The presence of a dimethylamino moiety ininhibitors JNK-IN-7, THZ531, THZ1, and neratinib, as shown in Table 3,led to the production of specific fragment ions not observed in theother probes.

TABLE 3 Ions Produced by MS/MS of Representative Modified Test-Compound-Polypeptide Conjugates THZ1; Target = CDK7 ION PROPOSED STRUCTUREFORMULA Ion m/z Test Compound

C31 H28 Cl1 N7 O2 N/A TH (Thiolated ion)

C31 H30 Cl1 N7 O2 S1 600.1943 (H+) RMA (Retro- Michael addition)

C31 H28 Cl1 N7 O2 566.2066 (H+) TH-DMAE (Thiolated ion withdimethylamine elimination)

C29 H23 Cl1 N6 O2 S1 555.1365 (H+) RMA- DMAE-1 (Retro- Michael addition/dimethyl amine elimination type 1)

C29 H23 Cl1 N6 O2 523.1644 (H+) RMA- DMAE-2 (Retro- Michael addition/dimethyl amine elimination type 2)

C29 H21 Cl1 N6 O2 521.1487 (H+) iy1

C25 H19 Cl1 N6 O1 455.1382 (H+) iy2

C18 H14 Cl1 N5 336.1011 (H+) ib2-RMA- DMAE-2 ib2 with retro-Michaeladdition and dimethylamine elimination type-2

C11 H8 N1 O2 186.0555 ib1

C6 H10 N1 O1 112.0762 THZ531; Target = CDK12 *, cleavage at alkylatedamine only observed at high collision energies PROPOSED STRUCTURE ION(1-9 corresponds to FIG. 2A) FORMULA Ion m/z Test Compound

(8) C30 H32 Cl1 N7 O2 H N/A TH (Thiolated ion)

(9) C30 H34 Cl1 N7 O2 S1 592.2256 (H+) RMA (Retro- Michael addition)

(8) C30 H32 Cl1 N7 O2 H 558.2379 (H+) TH-DMAE (Thiolated ion withdimethylamine elimination)

(7) C28 H27 Cl1 N6 O2 S1 547.1678 (H+) RMA- DMAE-1 (Retro- Michaeladdition/ dimethyl amine elimination type 1)

(6) C28 H27 Cl1N6 O2 515.1957 (H+) RMA- DMAE-2 (Retro- Michael addition/dimethyl amine elimination type 2)

(5) C28 H25 Cl1 N6 O2 513.1800 (H+) iy1

(4) C24 H23 Cl1 N6 O1 447.1695 (H+) iy2

(3) C17 H18 Cl1 N5 328.1323 (H+) Cleavage at alkylated amine

*see note above C12 H9 Cl1 N4 245.0589 (H+) ib2-RMA- DMAE-2 ib2 withretro- Michael addition and dimethylamine elimination type-2

(2) C11 H8 N1 O2 186.0555 ib1

(1) C6 H10 N1 O1 112.0762 QL-47; Target = BTK ION PROPOSED STRUCTUREFORMULA Ion m/z Compound modified immonium ion

C29 H26 N6 O2 S 523.1911 (H+) Test Compound

C27 H21 N5 O2 N/A TH (Thiolated ion)

C27 H23 N5 O2 S1 482.1645 (H+) TH-H2 (Thiolated ion with H2 elimination)

C27 H21 N5 O2 S1 480.1489 (H+) RMA (Retro- Michael addition)

C27 H21 N5 O2 448.1768 (H+) iy1

C24 H19 N5 O1 394.1662 (H+) Ibrutinib; Target = BTK ION PROPOSEDSTRUCTURE FORMULA Ion m/z Compound modified immonium ion

C27 H29 N7 O2 S 516.2176 (H+) Test Compound

(3) C25 H24 N6 O2 N/A TH (Thiolated ion)

(5) C25 H26 N6 O2 S1 475.1911 (H+) Thiolated ion with H2 elimination

(4) C25 H24 N6 O2 S1 473.1754 (H+) RMA (Retro- Michael addition)

(3) C25 H24 N6 O2 441.2034 (H+) iy1

(2) C22 H22 N6 O1 387.1928 (H+) Cleavage at alkylated amine

(1) C17 H13 N5 O1 304.1193 (H+) RMA with alkylated amine cleavage

C8 H12 N1 O1 138.0913 (H+) Neratinib; Target = EGFR/Her2 ION PROPOSEDSTRUCTURE FORMULA Ion m/z Test Compound

C30 H29 Cl1 N6 O3 N/A TH (Thiolated ion)

C30 H31 Cl1 N6 O3 S1 591.19396 (H+) RMA (Retro- Michael addition)

C30 H29 Cl1 N6 O3 557.20624 (H+) TH-DMAE (Thiolated ion withdimethylamine elimination)

C28 H24 Cl1 N5 O3 S1 546.13611 (H+) RMA- DMAE-1 (Retro- Michaeladdition/ dimethyl amine elimination type 1)

C28 H24 Cl1N5 O3 514.16404 (H+) RMA- DMAE-2 (Retro- Michael addition/dimethyl amine elimination type 2)

C28 H22 Cl1N5 O3 512.14839 (H+) iy1

C24 H20 Cl1 N5 O2 446.13783 (H+) ib1

C6 H10 N1 O1 112.07624 TL10-201; Target = JAK3 ION PROPOSED STRUCTUREFORMULA Ion m/z Compound modified immonium ion

C21 H25 Cl N8 O2 S 489.1582 (H+) Test Compound

C19 H20 Cl1N7 O2 N/A TH (Thiolated ion)

C19 H22 Cl1 N7 O2 S1 448.13170 (H+) Thiolated ion with H2 elimination

C19 H20 Cl1 N7 O2 S1 446.1160 (H+) RMA (Retro- Michael addition)

C19 H20 Cl1N7 O2 414.14398 (H+) iy1

C16 H18 Cl1 N7 O1 360.13341 (H+) Cleavage at alkylated amine

C9 H11 Cl1 N6 O1 255.07556 (H+) MI-2; Target = MALT1 ION PROPOSEDSTRUCTURE FORMULA Ion m/z Test Compound

C19 H17 Cl3 N4 O3 N/A TH (Thiolated ion)

C19 H18 Cl2 N4 O3 S1 453.05494 (H+) Thiolated ion with H2 elimination

C19 H16 Cl2 N4 O3 S1 451.03929 (H+) iy1

C17 H16 Cl2 N4 O2 379.07231 (H+) HBX-19818; Target = USP7 ION PROPOSEDSTRUCTURE FORMULA Ion m/z Compound modified immonium ion

C27 H32 N4 O S 461.2370 (H+) Test Compound

C25 H28 Cl1 N3 O1 N/A TH (Thiolated ion)

C25 H29 N3 O1 S1 420.21041 (H+) Cleavage at alkylated amine

C18 H23 N3 O1 S1 330.1635 (H+) Elimination product

C15 H14 N2 O1 S1 271.0900 (H+) ib1

C14 H12 N1 O1 S1 242.06396 ia1 (loss of CO from ib1)

C13 H12 N1 S1 214.06904

To explore the fragmentation pathways in the context of alternativewarheads and target families, HBX-19818 (a deubiquitinase (DUB)inhibitor, (see e.g., Reverdy et al, Chem. Biol. 2012, 19, 467-477) aswell as the paracaspase inhibitor MI-2 (see e.g., Fontan et al, CancerCell, 2012, 22:812-824) were investigated (see e.g., Table 1 and Table3), both of which modify cysteine residues. Thiolated ions were observedin the MS/MS spectra of peptides labeled with each probe, confirmingthis as a dissociation pathway shared across covalent inhibitors whichmodify their targets through a thioether bond (see e.g., FIGS. 8D, 8F,and 8H). Consistent with the data above, amide bond cleavage wasobserved within each inhibitor (see e.g., FIG. 8G, labeled “1” and FIG.8H, labeled “1”). It was further observed that peptides covalentlymodified with HBX-19818 or MI-2 did not undergo retro-Michael addition,but rather a low-yield elimination reaction to produce a series ofdehydroalanine-containing b- and y-type fragment ions (see e.g., FIG.8F). It was determined that the dissociation pathways described aboveare agnostic with respect to peptide sequence, charge state, andproteolytic enzyme.

Collectively, these results suggest that probes which form covalentadducts through a thioether linkage dissociate under MS/MS conditions toyield predictable, structurally specific fragment ions.

Example 6. Spectral Match Scores

Though covalently modified peptides were identified as described inExample 5, in many instances the associated fragment ions providedrelatively low spectral match scores when using the commercial MASCOTalgorithm (see e.g., Perkins et al, Electrophoresis, 1999, 20:3551-3567)for database search and sequence assignment. For example, thehigh-resolution MS/MS spectrum shown in FIG. 2A (THZ531) yielded aconfidence peptide score of 15.68. A majority of kinase inhibitorscontain one or more heterocyclic rings that impart significant gas-phasebasicity, leading to increased peptide charge state (typically ≥3+,shown in FIGS. 9A and 9B). Dissociation of higher charge state peptidescan yield complicated product ion spectra containing multiply-chargedfragment ions which can diminish the performance of search algorithms.To account for these effects the spectral pre-processing scripts weremodified to normalize all fragment ions to the 1+ charge state. Thismodification increased the MASCOT score for the MS/MS spectrum to 40.

Without being bound by theory, it was surmised that the myriad of MS/MSions derived from fragmentation of the inhibitor (see e.g. FIG. 2B andTables 2-3) further diminished the quality of spectral matches. To testthis hypothesis MS/MS spectra were pre-processed (e.g., prior tosubmission for MASCOT database search) based on the fragmentationpathways described above in Example 5. First, neutral loss of theinhibitor from the peptide backbone was defined as part of thevariable/fixed modification in MASCOT; this step increased the peptidescore to 50. Second, the remaining inhibitor-related ions described inTables 2-3 were removed (FIG. 2B), which further improved the MASCOTscore to 66, as shown in FIG. 2C. To assess these improvements across alarger population of modified peptides, THZ531 and THZ-1 with reducedbovine serum albumin (BSA) and the MASCOT peptide scores were comparedfor spectra which were subject to minimal (deisotope only) or extensivepre-processing. It was observed that use of the full pre-processingscheme as described above led to consistent and significant improvementsin MASCOT scores for >90% of MS/MS spectra, as shown in FIG. 2D. Theseresults demonstrate that the consistent, inhibitor-associatedfragmentation pathways can be used to improve the ability to identifycovalently modified peptides.

Example 7. Targeted Mass Spectrometry

Based on the results described in Example 5-6, it was speculated thatinhibitor-associated fragmentation pathways can be used as the basis forhighly-selective precursor scanning or other targeted mass spectrometryassays. As a prerequisite to these experiments, the yield ofinhibitor-specific fragment ions as a function of kinetic energy duringMS/MS was investigated. Accordingly, conjugated modelcysteine-containing peptides with 8 different acrylamide inhibitors wereprepared. Next, direct infusion of the mixture was used to acquire MS/MSspectra for each modified peptide across a range of collision energies(CE). Plots of ion yield as a function of CE revealed several trends.For example, it was observed that use of higher CE resulted in gas phaseenrichment of structure-specific fragment ions from each inhibitorrelative to canonical peptide b- and y-type ions. In fact, the iy_(n)ions reached a maximum yield at >40 eV, a CE where peptide fragment ionintensities were greatly reduced, as shown in FIGS. 3A-3C and FIGS.10A-10D. In addition, for specific ion types (e.g., thiolated ion) thereexisted sufficient overlap in CE profiles such that a single collisionenergy generated significant signal intensity (>50% max) for at leastone charge state of a given peptide, as shown in FIGS. 10E and 10F.Finally, inhibitors incorporating a dimethylamino group produced acharacteristic fragment at m/z=112, a relatively ‘quiet’ region ofpeptide MS/MS spectra, as shown in FIG. 10D.

Next the energetics associated with inhibitor-specific ions wasinvestigated to develop precursor scan mass spectrometry methods toenable selective detection of peptide-probe conjugates. Two peptideswere labeled with ibrutinib and spiked into a complex mixture of trypticpeptides derived from human myeloid K562 cells. Precursor scans werethen performed on a triple quadrupole mass spectrometer using thecollision energies as described above. FIGS. 4A-4G and FIGS. 11A-11Fshow that selective detection of the thiolated (FIG. 4F) or alkylatedamine cleavage product (m/z=304.12, FIG. 11F) ions provide more than anorder of magnitude improvement in selectivity as compared to standardfull mass range data acquisition (FIG. 4C and FIG. 11C). As a furthertest of selectivity we next triggered full-scan MS/MS acquisition basedon precursor ion signals corresponding to Ibrutinib thiolated ions (m/z475.19; FIG. 4D). After pre-processing of the resulting peak lists, aMASCOT search yielded 22 peptide-spectral matches (PSMs), correspondingto 16 unique peptide sequences. Notably, the spiked-in synthetic was theonly Ibrutinib-modified peptide detected (FIG. 4G, MASCOT score ˜49),even though it was amongst the lowest intensity precursor ions in thebase peak chromatogram (BPC). Similar gas phase enrichment was observedfor precursor ion data obtained for QL47 (targeting thiolated ion andiy₁, shown in FIG. 12A-12J). These data suggest that predictableinhibitor-associated fragmentation may be used as a basis to developselective mass spectrometry assays for detection of proteins targeted bycysteine-directed covalent probes.

Several novel dissociation pathways were identified based on probestructure and reactive warhead (i.e., reactive group). Our resultsprovide evidence for several informative trends: (i) Probes covalentlybound by a thioether linkage produce a characteristic thiolated ionindependent of peptide sequence and charge state; (ii) Certaindissociation pathways appear to be class-specific; for example,acrylamide warheads undergo a retro Michael addition to regenerate theintact probe, while inhibitors with dimethylamino groups yield alow-mass fragment at m/z=112; (iii) Information for these novelfragmentation pathways can be used to markedly improve peptide sequenceidentification scores for shotgun proteomic methods; (iv) Use of higherCE provides gas phase enrichment of inhibitor-specific fragmentsrelative to canonical peptide b- and y-type ions.

Example 8. Target Engagement Stoichiometry Assay

HeLa-S3 cells were treated with 100 nM THZ1 (covalent probe that targetscys-312 on the kinase CDK7) or DMSO (control) for 6 hours. CDK7 wasenriched from total cell extracts by immuno-precipitation. The resultingtreated (THZ1) and control (DMSO) immuno-precipitates were alkylatedwith heavy or light N-(2,5-dimethylphenyl)-2-iodoacetamide (DMPIA),respectively. Samples were combined, digested with trypsin, anddesalted. The LC-MS/MS method comprised acquisition of targeted MS/MSspectra for both the light and heavy DMPIA-labeled CDK7 peptidescontaining cys-312: Y-F-S-N-R-P-G-P-T-P-G-C*Q-L-P-R-P-N-C*P-V-E-T-L-K(SEQ ID NO: 11; C* refers to a cysteine residue labeled with DMPIA). Inaddition, a MS/MS collision energy of 90 eV was used to simultaneouslymaximize the yield of thiolated ions, or derivative ions thereof, whileminimizing the signals for typical fragment ions resulting from peptideamide bond cleavages. Collision energy profiles for the peptides labeledwith the broad thiol-reactive reagents are shown in FIG. 5 .Quantification of the CDK7 target engagement in shown in FIG. 6 . Arepresentative diagram visualizing the workflow of the target engagementstoichiometry assay in shown in FIG. 7 .

Table 4 shows a list of broad thiol reactive reagents tested in thetarget engagement stoichiometry assay showing the chemical structure andchemical formula of the parent sample compounds and related fragmentions. The chemical structure, chemical formula and mass to charge ratio(m/z) for derivative fragment ions produced in the gas phase duringMS/MS is also shown.

TABLE 4 Reagents and Related Fragment Ions ION PROPOSED STRUCTUREFORMULA ION m/z Parent Iodoacetamide 2-iodo-1- morpholinoethan-1- one

C6H10INO2 N/A Compound- modified immonium

C8H15N2O2S+ 203.08487 Thiolated

C6H12NO2S+ 162.05833 Thiolated-H2

C6H10NO2S+ 160.04268 Thiolated-H2-CO

C5H10NOS+ 132.04776 Parent Iodoacetamide 1-((2R,6R)-2,6-dimethylmorpholino)- 2-iodoethan-1-one

C8H14INO2 N/A Compound-modified immonium

C10H19N2O2S+ 231.11617 Thiolated

C8H16NO2S+ 190.08963 Thiolated-H2

C8H14NO2S+ 188.07398 Thiolated-H2-CO

C7H14NOS+ 160.07906 Parent Iodoacetamide 1-((2R,6S)-2,6-dimethylmorpholino)- 2-iodoethan-1-one

C8H14INO2 N/A Compound- modified immonium

C10H19N2O2S+ 231.11617 Thiolated

C8H16NO2S+ 190.08963 Thiolated-H2

C8H14NO2S+ 188.07398 Thiolated-H2-CO

C7H14NOS+ 160.07906 Parent Iodoacetamide 1-(2,2- dimethylmorpholino)-2-iodoethan-1-one

C8H14INO2 N/A Compound-modified immonium

C10H19N2O2S+ 231.11617 Thiolated

C8H16NO2S+ 190.08963 Thiolated-H2

C8H14NO2S+ 188.07398 Thiolated-H2-CO

C7H14NOS+ 160.07906 Parent Iodoacetamide 1-(3,5- dimethylmorpholino)-2-iodoethan-1-one

C8H14INO2 N/A Compound-modified immonium

C10H19N2O2S+ 231.11617 Thiolated

C8H16NO2S+ 190.08963 Thiolated-H2

C8H14NO2S+ 188.07398 Thiolated-H2-CO

C7H14NOS+ 160.07906 Parent Iodoacetamide 1-(hexahydrocyclopenta[b][1,4]oxazin- 4(4aH)-yl)-2- iodoethan-l-one

C9H14INO2 N/A Compound-modified immonium

C11H19N2O2S+ 243.11617 Thiolated

C9H16NO2S+ 202.08963 Thiolated-H2

C9H14NO2S+ 200.07398 Thiolated-H2-CO

C8H14NOS+ 172.07906 Parent Iodoacetamide 1-(2,3-dihydro-4H-benzo[b][1,4]oxazin- 4-yl)-2-iodoethan- 1-one

C10H10INO2 N/A Compound-modified immonium

C12H15N2O2S+ 251.08487 Thiolated

C10H12NO2S+ 210.05833 Thiolated-H2

C10H10NO2S+ 208.04268 Cyclic-Thiolated- H2-SH2

C10H8NO2+ 174.05495 Parent Iodoacetamide N-(4-cyanophenyl)-2-iodoacetamide

C9H7IN2O N/A Compound- modified immonium

C11H12N3OS+ 234.06956 Thiolated

C9H9N2OS+ 193.04301 Thiolated-H2

C9H7N2OS+ 191.02736 Cyclic-Thiolated- H2-SH2

C9H5N2O+ 157.03964 Cyclic-Thiolated- H2-SH2-CO Structure to bedetermined C8H5N2+ 129.04472 Parent Iodoacetamide N-(2,5-dimethylphenyl)-2- iodoacetamide

C10H12INO N/A Compound-modified immonium

C12H17N2OS+ 237.10561 Thiolated

C10H14NOS+ 196.07906 Thiolated-H2

C10H12NOS+ 194.06341 Cyclic-Thiolated-H2-SH2

C10H10NO+ 160.07569 Parent Iodoacetamide N-(2,5- dimethoxyphenyl)-2-iodoacetamide

C10H12INO3 N/A Compound- modified immonium

C12H17N2O3S+ 269.09544 Thiolated

C10H14NO3S+ 228.06889 Thiolated-H2

C10H12NO3S+ 226.05324 Cyclic-Thiolated- H2-SH2

C10H10NO3+ 192.06552 Cyclic-Thiolated-H2- SH2-C3H2O Structure to bedetermined C7H8NO2+ 138.05495 Parent Iodoacetamide 2-iodo-N-phenylacetamide

C8H8INO N/A Compound- modified immonium

C10H13N2OS+ 209.07431 Thiolated

C8H10NOS+ 168.04776 Thiolated-H2

C8H8NOS+ 166.03211 Cyclic-Thiolated- H2-SH2

C8H6NO+ 132.04439 Parent Iodoacetamide 2-iodo-N-(p- tolyl)acetamide

C9H10INO N/A Compound- modified immonium

C11H15N2OS+ 223.08996 Thiolated

C9H12NOS+ 182.06341 Thiolated-H2

C9H10NOS+ 180.04776 Cyclic-Thiolated- H2-SH2

C9H8NO+ 146.06004 Parent Iodoacetamide 2-iodo-1-(4- methylpiperazin-1-yl)ethan-1-one 2,2,2- trifluoroacetate

C9H14F3IN2O3 N/A Compound- modified immonium

C9H18N3OS+ 216.11651 Thiolated

C7H15N2OS+ 175.08996 Thiolated-H2

C7H13N2OS+ 173.07431 Thiolated-H2-SH2

C5H13N2+ 101.10732 Parent Iodoacetamide N-(furan-2- ylmethyl)-2-iodoacetamide

C7H8INO2 N/A Compound- modified immonium

C9H13N2O2S+ 213.06922 Parent Iodoacetamide 2-iodo-N-(1-methyl-1H-imidazol-4- yl)acetamide

C6H8IN3O N/A Compound-modified immonium

C8H13N4OS+ 213.08046 Compound-modified immonium-NH3

C8H10N3OS+ 196.05391 Thiolated

C6H10N3OS+ 172.05391 Thiolated-H2

C6H8N3OS+ 170.03826 Thiolated-H2O

C6H8N3S+ 154.04334 Thiolated-CH2S

C5H8N3O+ 126.06619

OTHER EMBODIMENTS

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

What is claimed is:
 1. An analytical method, comprising: i) contacting abroad thiol reactive compound with a polypeptide to form a broad thiolreactive compound-polypeptide conjugate; ii) analyzing the broad thiolreactive compound-polypeptide conjugate using a mass spectrometry assay;iii) detecting one or more thiolated ions of the broad thiol reactivecompound, or derivative ions thereof, produced in the mass spectrometryassay; and iv) identifying that the broad thiol reactive compound formsan irreversible bond with the polypeptide based on the detection of theone or more thiolated ions, or derivative ions thereof, in the massspectrometry assay; wherein the thiolated ions or derivatives thereofare fragment ions.
 2. The method of claim 1, wherein thecompound-polypeptide conjugate comprises one or more thioether bondsbetween the compound and the polypeptide.
 3. The method of claim 1,wherein step i) comprises contacting the compound and the polypeptide inthe presence of a first solvent component.
 4. The method of claim 1,wherein step i) further comprises contacting the compound and thepolypeptide in the presence of a buffer agent.
 5. The method of claim 1,wherein step i) is performed using a molar excess of the compoundcompared to the polypeptide.
 6. The method of claim 1, furthercomprising contacting the broad thiol reactive compound-polypeptideconjugate with an acid in the presence of a second solvent componentprior to performing the mass spectrometry assay of step ii).
 7. Themethod of claim 1, wherein the method further comprises digesting thebroad thiol reactive compound-polypeptide conjugate prior to theperforming the mass spectrometry assay of step ii).
 8. The method ofclaim 7, wherein the digesting comprises reacting the broad thiolreactive compound-polypeptide conjugate with trypsin in the presence ofa third solvent component.
 9. The method of claim 1, wherein thepolypeptide comprises one or more amino acids residues comprising atleast one sulfur atom.
 10. The method of claim 9, wherein thepolypeptide comprises one or more cysteine residues.
 11. The method ofclaim 10, wherein the broad thiol reactive compound is identified asirreversibly bonding to one or more cysteine residues of thepolypeptide.
 12. The method of claim 11, wherein the broad thiolreactive compound comprises one or more groups independently selectedfrom the group consisting of acrylamide groups, dimethylamino acrylamidegroups, iodoacetamide groups, chloroacetamide groups, maleimide groups,and C—X groups, wherein X is a halogen.
 13. The method of claim 1,wherein the broad thiol reactive compound is selected from the groupconsisting of 2-iodo-1-morpholinoethan-1-one;1-((2R,6R)-2,6-dimethylmorpholino)-2-iodoethan-1-one;1-((2R,6S)-2,6-dimethylmorpholino)-2-iodoethan-1-one;1-(2,2-dimethylmorpholino)-2-iodoethan-1-one;1-(3,5-dimethylmorpholino)-2-iodoethan-1-one;1-(hexahydrocyclopenta[b][1,4]oxazin-4(4aH)-yl)-2-iodoethan-1-one;1-(2,3-dihydro-4H-benzo[b][1,4]oxazin-4-yl)-2-iodoethan-1-one;N-(4-cyanophenyl)-2-iodoacetamide;N-(2,5-dimethylphenyl)-2-iodoacetamide;N-(2,5-dimethoxyphenyl)-2-iodoacetamide; 2-iodo-N-phenylacetamide;2-iodo-N-(p-tolyl)acetamide;2-iodo-1-(4-methylpiperazin-1-yl)ethan-1-one 2,2,2-trifluoroacetate;N-(furan-2-ylmethyl)-2-iodoacetamide;2-iodo-N-(1-methyl-1H-imidazol-4-yl)acetamide; and N-ethylmaleimide. 14.The method of claim 1, wherein the polypeptide is a protein or a proteinfragment.
 15. The method of claim 1, wherein the polypeptide is aprotein fragment comprising from about 10 to about 30 amino acidresidues.
 16. The method of claim 1, wherein the polypeptide is a kinaseor a fragment thereof, or a deubiquitinase or a fragment thereof. 17.The method of claim 1, wherein the polypeptide is a kinase selected fromthe group consisting of JNK2, JAK3, CDK7, CDK12, ITK, USP-7, TAK1, andEGFR, or a fragment thereof.
 18. The method of claim 1, wherein thepolypeptide is a kinase fragment comprising from about 10 to about 30amino acid residues.
 19. The method of claim 1, wherein the polypeptidecomprises an amino acid sequence having at least 90% sequence identityto a sequence selected from the group consisting of: (SEQ ID NO: 1)L-M-D-A-N-L-C-Q-V-I-Q-M-E; (SEQ ID NO: 2) L-V-M-E-Y-L-P-S-G-C-L-R;(SEQ ID NO: 3) M-A-P-P-D-L-P-H-W-Q-D-C-H-E-L-W-S-K; (SEQ ID NO: 4)H-G-C-L-S-D-Y-L-R-S-Q-R-G-L-F-A-A-E; (SEQ ID NO: 5)Y-F-S-N-R-P-G-P-T-P-G-C-Q-L-P-R-P-N-C-P-V-E-T-L-K; (SEQ ID NO: 6)G-C-L-L-D-Y-V-R; (SEQ ID NO: 7) F-G-L-C-S-G-P-A-D-T-G-R; (SEQ ID NO: 8)Y-M-A-N-G-C-L-sL-N-Y-L-R; (SEQ ID NO: 9)I-C-D-F-G-T-A-C-D-I-Q-T-H-M-T-N-N-K;  and (SEQ ID NO: 10)Y-F-S-N-R-P-G-P-T-P-G-C-Q-L-P-(13C6-15N4)R-P-N-C- P-V-E-T-L-K.